Antiviral purine compounds

ABSTRACT

Compounds of formula (I), and pharmaceutically acceptable salts thereof ##STR1## wherein R 1  is hydroxy, amino, chloro or OR 7  wherein 
     R 7  is C 1-6  alkyl, phenyl or phenyl C 1-2  alkyl either of which phenyl moieties may be substituted by one or two substituents selected from halo, C 1-4  alkyl or C 1-4  alkoxy; 
     R 2  is amino or, when R 1  is hydroxy or amino, R 2  may also be hydrogen; 
     R 3  is hydrogen, hydroxymethyl or acyloxymethyl; 
     R 4  is a group of formula: ##STR2## R 5  and R 6  are independently selected from hydrogen, C 1-6  alkyl and optionally substituted phenyl; or 
     R 3  and R 4  together are: ##STR3##  wherein R 6  is as defined above; 
     having antiviral activity, to processes for their preparation and their use as pharmaceuticals.

This application is a continuation of Ser. No. 530,736 (filed May 19,1990), abandoned, which in turn is a continuation-in-part of Ser. No.276,868 (filed Nov. 28, 1988), abandoned, of Ser. No. 387,068 (filedJul. 28, 1989), U.S. Pat. No. 5,055,458, and of Ser. No. 528,575 (filedMay 19, 1990) U.S. Pat. No. 5,108,994.

The present invention relates to compounds having antiviral activity, toprocesses for their preparation and to their use as pharmaceuticals.

EP-A-242482 (Beecham Group p.l.c.) describes a group of guaninederivatives having a 9-hydroxyalkoxy substituent, and possessingantiviral activity.

A novel, structurally distinct class of compounds has now beendiscovered, these compounds also having antiviral activity.

Accordingly, the present invention provides a compound of formula (I),or a pharmaceutically acceptable salt thereof: ##STR4## wherein

R₁ is hydroxy, amino, chloro or OR₇ wherein

R₇ is C₁₋₆ alkyl, phenyl or phenyl C₁₋₂ alkyl either of which phenylmoieties may be substituted by one or two substituents selected fromhalo, C₁₋₄ alkyl or C₁₋₄ alkoxy;

R₂ is amino or, when R₁ is hydroxy or amino, R₂ may also be hydrogen;

R₃ is hydrogen, hydroxymethyl or acyloxymethyl;

R₄ is a group of formula: ##STR5## wherein

R₅ and R₆ are independently selected from hydrogen, C₁₋₆ alkyl andoptionally substituted phenyl; or

R₃ and R₄ together are: ##STR6## wherein

R₆ is as defined above.

When R₁ is hydroxy and R₂ is amino, the compound of formula (I) is aguanine derivative;

When R₁ is amino and R₂ is hydrogen, the compound of formula (I) is anadenine derivative;

When R₁ is hydroxy and R₂ is hydrogen, the compound of formula (I) is ahypoxanthine derivative; and

When R₁ and R₂ are both amino groups, the compound of formula (I) is a2,6-diaminopurine derivative.

Often, the compound of formula (I) is a guanine or adenine derivative,preferably an adenine derivative.

Suitable examples of the acyl group in R₃ when acyloxymethyl, includecarboxylic acyl, such as C₁₋₇ alkanoyl and benzoyl optionallysubstituted in the phenyl ring include acetyl, propionyl, butyryl,heptanoyl and hexanoyl.

Suitable examples of R₅ and R₆ include hydrogen, methyl, ethyl, n- andiso-propyl, n-, sec-, iso- and tert-butyl, and phenyl optionallysubstituted by one, two or three groups or atoms selected from halogen,such as fluoro, chloro, bromo, and C₁₋₄ alkyl or C₁₋₄ alkoxy wherein thealkyl moiety is selected from those listed for R₅ /R₆ above.

Examples of R₇ include methyl, ethyl, n- and iso-propyl, phenyl andbenzyl optionally substituted by one or two of methyl, ethyl, n- andiso-propyl, methoxy, ethoxy, n- and iso-propoxy, fluoro, chloro orbromo.

Examples of pharmaceutically acceptable salts of the compound of formula(I) are acid addition salts formed with a pharmaceutically acceptableacid such as hydrochloric acid, orthophosphoric acid and sulphuric acid.Pharmaceutically acceptable salts also include those formed with organicbases, preferably with amines, such as ethanolamines or diamines; andalkali metals, such as sodium and potassium.

As the compound of formula (I) contains a phosphonate group, suitablesalts include metal salts, such as alkali metal salts, for examplesodium or potassium, alkaline earth metal salts such as calcium ormagnesium and ammonium or substituted ammonium salts, for example thosewith lower alkylamines such as triethylamine, hydroxy-lower alkylaminessuch as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine ortris-(2-hydroxyethyl)-amine.

It will be appreciated that some of the compounds of formula (I),especially those wherein R₃ is other than hydrogen, have an asymmetriccentre, and therefore are capable of existing in more than onestereoisomeric form. The invention extends to each of these formsindividually and to mixtures thereof, including racemates. The isomersmay be separated conventionally by chromatographic methods or using aresolving agent. Alternatively, the individual isomers may be preparedby asymmetric synthesis using chiral intermediates.

The compounds of formula (I) including their alkali metal salts may formsolvates such as hydrates and these are included wherever a compound offormula (I) or a salt thereof is herein referred to.

It will be appreciated that, when R₁ is hydroxy in formula (I) thecompound exists in the predominant tautomeric form of structure (IA):##STR7##

The invention also provides a process for the preparation of a compoundof formula (I), or a pharmaceuticallty acceptable salt thereof, whichprocess comprises either

i) imidazole ring closure of a compound of formula (II): ##STR8##wherein X is a group capable of cyclising to form an imidazole ring,such as amino or an amino derivative, for example, formylamino; or

ii) pyrimidine ring closure of a compound of formula (III): ##STR9##wherein Y is amino or C₁₋₆ alkoxy, with a condensing agent capable ofcyclising to form a pyrimidine ring having a 2-R₂ ' substituent, to givea compound of formula (I) wherein R₁ is hydroxy and R₂ is amino; or

iii) condensing a compound of formula (IV): ##STR10## with a side chainintermediate of formula (V):

    ZCH.sub.2 --CHR.sub.3 'OR.sub.4 '                          (V)

wherein Z is a leaving group;

and wherein, in formulae (II) to (V), R₁ ', R₂ ', R₃ ', R₄ ' are R₁, R₂,R₃ and R₄ respectively, or groups or atoms convertible thereto; andthereafter, when desired or necessary, converting R₁ ', R₂ ', R₃ 'and/or R₄ ', when other than R₁, R₂, R₃ and/or R₄ to R₁, R₂, R₃ and/orR₄ respectively, and/or converting R₁ ', R₂ ', R₃ ' and/or R₄ ' when R₁,R₂, R₃ and/or R₄, to other R₁, R₂, R₃ and/or R₄.

Process i) may be carried out, preferably when X is formyl, using acyclisation condensing agent, such as diethoxymethyl acetate or triethylorthoformate, or by fusion.

Process ii) is preferably carried out in accordance with the methodsdescribed in EP-A-242482, the subject matter of which is incorporatedherein by reference.

Process iii) may be carried out with suitable values for Z includinghydroxy and halo, such as chloro, bromo and iodo, preferably iodo; orother groups readily displaceable by nucleophiles, such as mesyloxy ortosyloxy. The reaction preferably takes place in an inert solvent, suchas dimethylformamide at 0°-50° C., preferably ambient temperature. WhenZ is hydroxy, the reaction takes place in the presence of a dehydratingcatalyst, such as diethyl azodicarboxylate in the presence oftriphenylphosphine. When Z is halo, the reaction preferably takes placein the presence of a base, such as potassium carbonate.

Examples of conversions of variable groups are as follows:

R₁ '--R₁

a) An R₁ hydroxy group may be converted to R₁ ' is chloro, bychlorination using a reagent such as phosphorus oxychloride, preferablyin the presence of tetraethylammonium chloride and dimethylaniline (asacid acceptor) in CH₃ CN at reflux temperatures, according to the methoddescribed by M. J. Robins and B. Ozanski Can. J. Chem, 59, 2601 (1981).

b) An R₁ ' chloro group may be converted to R₁ is hydroxy by hydrolysisusing aqueous mineral acid, such as hydrochloric acid, or morepreferably, using an organic acid, such as formic acid at elevatedtemperature, suitably 70°-150° C., preferably around 100° C.

c) An R₁ ' chloro group may be converted to R₁ is amino by treatmentwith ammonia in a lower alkanol, such as ethanol or methanol in anautoclave at 100° C. for a period of about 7 hours, or alternatively, bytreatment with sodium azide in dimethylformamide (forming an R₁ is N₃intermediate), followed by reduction with ammonium formate/palladium orcharcoal, in methanol.

d) An R₁ ' alkoxy group, such as methoxy, may be converted to R₁ hydroxyby the methods of D. R. Haines, J. Med. Chem. 1987, 30, 943 and K. K.Ogilvie and H. R. Hanna, Can. J. Chem. 1984, 62, 2702.

e) An R₁ ' protected amino group, such as tritylamino, may be convertedto amino, by treatment with aqueous acetic acid, preferably 80% aceticacid at elevated temperature, around 80° C. R₁ ' may also bephthalimido, which may be converted to amino by treatment with methylhydrazine or hydrazine in an inert solvent, such as dichloromethane, atambient temperature.

R₂ '--R₂

a) R₂ may be protected amino, such as formylamino, which may beconverted to R₂ is amino by hydrolysis.

R₃ '--R₃

a) R₃ hydroxymethyl may be converted to R₃ acyloxymethyl by conventionalacylation procedures.

b) R₃ ' may be protected hydroxymethyl, which may be converted to R₃hydroxymethyl by conventional deprotection methods.

Suitable examples of protecting groups and their removal, are asdescribed in EP-A-242482. Particularly suitable protecting groupsinclude the benzyl group, removed by catalytic hydrogenation usingpalladium/charcoal, 80% acetic acid; the acetate group removed by acidhydrolysis, 2M HCl in ethanol; or the t-butyldimethylsilyl groupremovable by 80% acetic acid at elevated temperature, around 90° C.

R₄ '--R₄

a) When R₅ and R₆ in R₄ are other than hydrogen, they may be convertedto R₅ and R₆ are hydrogen, using a deesterifying reagent, such astrimethylsilyl bromide in an aprotic solvent such as dichloromethane ordimethylformamide at ambient temperature, as described by C. E. McKennaet. al. J.C.S. Chem. Comm., 1979, 739.

Selective conversion of one R₅ and R₆ to hydrogen, may be achieved bytreatment with hydroxide ion, as described by Rabinowitz JACS 1960, 82,4564.

b) R₄ ' may be hydrogen, which may be converted to R₄, by treatment withQR₄ wherein Q is a leaving group and R₄ is as defined. Q is preferably atosyloxy group. Conditions for this reaction are i) preliminaryformation of an alkoxide using a base, such as sodium hydride, in anaprotic solvent for example dimethylformamide ii) reaction with QR₄ ataround ambient temperature. The reaction is as described A. Holy et. al.Collect. Czech. Chem. Comm. 1982, 47, 3447.

In this case, R₅ and R₆ are preferably other than hydrogen.

c) R₄ ' may be hydrogen, which may be converted, when R₃ ishydroxymethyl, to R₄ is CH₂ PO(OH)(OR₅), by treatment with ClCH₂ PCl₂followed by treatment with a base, followed by OR₅ --, according to themethod described by A. Holy et. al. Czech. Chem. Comm. 1985, 50, 1507;ibid 1987, 52. 2775.

It will be appreciated that the above conversions may take place in anydesired or necesssary order, having regard to the final desired compoundof formula (I).

Intermediates of formula (II) may be prepared from a correspondingcompound of formula (VI): ##STR11## and via intermediates of formula (V)wherein Z is OH, as hereinbefore defined, according to the methodsdescribed in EP-A-242482 i.e. by converting the compound of formula (V)wherein Z is OH to the phthalimidooxy derivative followed by reactionwith methylhydrazine, as described in Descriptions 1 and 4 hereinafter.

The compound of formula (VI) wherein R₁ is chloro and R₂ is amino, is aknown compound as described by Temple et. al. J. Org. Chem., 40 (21),3141, 1975.

The compound of formula (VI) wherein R₁ is chloro and R₂ is hydrogen isa commercially available compound.

Intermediates of formula (III) may be prepared according to the methodsdescribed in EP-A-242482.

Compounds of the formula (IV) are prepared from compounds of formula(VI) wherein the 5-amino group is formulated by reaction with R₉ ONH₂wherein R₉ is a protecting group, to give a compound of formula (VII):##STR12## which may be cyclised with diethoxymethyl acetate, to give acompound of formula (IV) wherein the OH group is protected. Suitablevalues for R₉ include benzyl, removable by hydrogenation, and thetetrahydropyran-2-yl group removable by treatment with 80% acetic acid,at ambient temperature.

Intermediates of the formula (V) wherein Z is hydroxy are knowncompounds or are prepared by analogous methods to those used forstructurally similar known compounds.

It will be appreciated that, when R₃ is hydroxymethyl or acyloxymethylin the resulting compound of formula (I), synthesis of the intermediateof formula (V) wherein Z is hydroxy may involve selective deprotectionof an intermediate wherein Z is protected hydroxy and R₃ is protectedhydroxymethyl, for example, as described in Description 4(b)hereinafter.

Intermediates of formulae (II), (III) and (V) but wherein Z is replacedby an aminooxy group, and wherein R₄ ' is R₄ as defined in formula (I),are believed to be novel and form an aspect of the invention.

Intermediates of the formula (IV) wherein R₁ ' is chloro and R₂ ' ishydrogen are also believed to be novel and form an aspect of theinvention.

Pharmaceutically acceptable salts may be prepared in conventionalmanner, for example, in the case of acid addition salts, by reactionwith the appropriate organic or inorganic acid.

It will be appreciated that the invention provides a process for thepreparation of a compound of formula (I) wherein R₃ is hydroxymethylwhich process comprises the deprotection of a compound of formula (I)wherein an OH group in R₃ is in protected form. Preferred methods fordeprotection, as hereinbefore described include removal of the benzyl,acetate or tbutyldimethylsilyl group.

The invention also provides a process for the preparation of a compoundof formula (I) wherein R₅ and R₆ are both hydrogen, which processcomprises the deesterification of a corresponding compound of formula(I) wherein R₅ and R₆ are the same alkyl or optionally substitutedphenyl group.

The compounds of the invention are of potential use in the treatment ofinfections caused by viruses, especially herpesviruses such as herpessimplex type 1, herpes simplex type 2, varicella-zoster virus,Epstein-Barr virus and cytomegalovirus; and lentiviruses such as visnavirus and human immunodeficiency virus (HIV 1 and 2)

The compounds may also be inhibitors of tumorogenic viruses and/or ofpotential use in the treatment of neoplastic diseases, i.e. cancer.

Compounds of the invention may be formulated for use in a pharmaceuticalcomposition. Accordingly, in a further aspect of the invention, there isprovided a pharmaceutical composition which comprises a compound offormula (I) or pharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier or excipient.

A composition which may be administered by the oral route to humans maybe compounded in the form of a syrup, tablet or capsule. When thecomposition is in the form of a tablet, any pharmaceutical carriersuitable for formulating such solid compositions may be used, forexample magnesium stearate, starch, lactose, glucose, rice, flour andchalk. The composition may also be in the form of an ingestible capsule,for example of gelatin, to contain the compound, or in the form of asyrup, a solution or a suspension. Suitable liquid pharmaceuticalcarriers include ethyl alcohol, glycerine, saline and water to whichflavouring or colouring agents may be added to form syrups. Thecompounds may also be presented with a sterile liquid carrier forinjection.

The composition may also be formulated for topical application to theskin or eyes.

For topical application to the skin, the composition may be in the formof a cream, lotion or ointment. These formulations may be conventionalformulations well known in the art, for example, as described instandard books of pharmaceutics and cosmetics, such as Harry'sCosmeticology published by Leonard Hill Books and the BritishPharmacopaeia.

The composition for application to the eyes may be a conventionaleye-drop composition well known in the art, or an ointment composition.

Preferably, the composition of this invention is in unit dosage form orin some other form that may be administered in a single dose. A suitabledosage unit might contain from 50 mg to 1 g of active ingredient, forexample 100 to 500 mg.

Such doses may be administered 1 to 4 times a day or more usually 2 or 3times a day. The effective dose of compound will in general be in therange of from 1.0 to 20 mg/kg of body weight per day or more usually 2.0to 10 mg/kg per day.

No unacceptable toxicological effects are indicated at the abovedescribed dosage levels.

The invention also provides a method of treating viral infections in ahuman or non-human animal, which comprises administering to the animalan effective, non-toxic amount of a compound of formula (I) or apharmaceutically acceptable salt thereof.

The invention also provides a compound of formula (I) or apharmaceutically acceptable salt thereof for use as an activetherapeutic substance, in particular for the treatment of viralinfections.

The compounds of the invention are also believed to exhibit asynergistic antiviral effect in conjunction with interferons; andcombination products comprising these two components for sequential orconcomitant administration, by the same or different routes, aretherefore within the ambit of the present invention. The followingexamples illustrate the invention; the following descriptions illustratethe preparation of intermediates.

Description 1 Intermediates (V) for Examples 1 to 4

(a) Diethyl 2-hydroxyethoxymethylphosphonate

To a solution of diethyl 2-acetoxyethoxymethylphosphonate* (16 g, 63mmol) in absolute ethanol (150 ml), was added 0.5M sodium ethoxide(12.19 ml). After standing at ambient temperature overnight, 1R-120Hresin was added until pH 6.5 was reached. The solution was filteredimmediately, the resin washed with ethanol and the combined filtratesevaporated under reduced pressure. The residue was chromatographed onsilica gel (eluted with chloroform: methanol, 98:2) to yield the titlecompound as a colourless oil (9.3g, 70%). IR: ν_(max) (film) 3420, 3000,1450, 1395, 1240, 1170, 1130, 1030, 970 cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃)1.35 (6H,t,J=7Hz, (OCH₂ CH₃)₂), 3.75 (4H,br.s, OCH₂ CH₂ O), 3.90(2H,d,J=8Hz, O--CH₂ P), 4.20 (4H,m,(O--CH₂ CH₃)₂), 5.20 (1H,br.s, D.sub.2 O exchangeable, OH). Found: C,38.64; H,8.27%. C₇ H₁₇ PO₅.0.3H₂ Orequires: C,38.63; H,8.01%.

(b) Diethyl 2-(N-phthalimidooxy)ethoxymethylphosphonate

Diethyl azodicarboxylate (8.12 g, 46.7 mmol) was added to a solution ofdiethyl 2-hydroxyethoxymethylphosphonate (9.0 g, 42.4 mmol),N-hydroxyphthalimide (6.92 g, 42.4 mmol) and triphenylphosphine (21.33g, 46.7 mmol) in dry tetrahydrofuran (150 ml) at ambient temperature,under a nitrogen atmosphere. After 3 days, the mixture was evaporated todryness, the residue dissolved in diethyl ether and the solution kept at4° C. for 7 days. The cooled mixture was filtered and the filtrateevaporated to dryness. The residue was chromatographed on silica gel(eluted with hexane: ethyl acetate, 50:50 *Can.J.Chem.60, 547(1982);Chem. Abs. 55. 15507c 1961 to remove triphenylphosphine oxide, thenchanged to ethyl acetate) to give the title compound as a light orangecoloured oil (10.6 g, 70%). IR: ν_(max) (film) 3510, 3000, 2950, 2920,1795, 1745, 1735, 1430, 1380, 1250, 1190, 1165, 1130, 1030, 980, 880,710 cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃) 1.32 (6H,t,J=7Hz, (--OCH₂ CH₃)₂), 3.93(2H,d, J=8Hz, O--CH₂ P), 3.98 (2H,m,CH₂ CH₂ OCH₂ P), 4.16 (4H,m, (OCH₂CH₃)₂), 4.39 (2H,m,N--OCH₂ CH₂), 7.74-786 (4H,m,phthalyl H). Found:C,50.65; H,5.94; N,3.76%. C₁₅ H₂₀ NO₇ P requires: C,50.42; H,5.64;N,3.92%. Found: m/z 358.1042(MH⁺). C15H₂₁ NO₇ P requires: m/z 358.1056.

c) Diethyl 2-(aminooxy)ethoxymethylphosphonate

To a solution of diethyl 2-(N-phthalimidooxy)ethoxymethylphosphonate(10.25 g, 29 mmol) in dry dichloromethane (55 ml) was addedmethylhydrazine (1.83ml, 34.4 mmol). After stirring at ambienttemperature for 2 hours, the reaction mixture was filtered andevaporated to dryness. The residue was chromatographed on silica gel(eluted with dichloromethane: methanol 98:2) to give the title compoundas an oil (6 g, 92%). IR: ν_(max) (film) 3480, 3320, 2995, 2920, 1600,1445, 1390, 1370, 1240, 1170, 970 cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃) 1.35(6H,t,J=7Hz, (OCH₂ CH₃)2), 3.8-3.95 (4H,m, O--CH₂ CH₂ O), 3.84(2H,d,J=8Hz, OCH₂ P), 4.21 (4H,dq,J=7Hz, 7Hz, (OCH₂ CH₃)2), 4.8(2H,br.s, D₂ O exchangeable, NH₂). Found: C,36.58; H,7.98; N,6.22%. C₇H₁₈ NO₅ P requires: C,37.00; H,7.98; N,6.17%. Found: m/z 228.0987(MH⁺).C₇ H₁₉ NO₅ P requires: m/z 228.1002.

Description 2 Intermediates for Examples 1 and 2

a)4-Chloro-6-2-(diethoxyphosphorylmethoxy)ethoxyamino]-5-formamidopyrimidine

A mixture of 4,6-dichloro-5-formamidopyrimidine (1.9 g, 10 mmol),diethyl 2-(aminooxy)ethoxymethylphosphonate (2.27 g, 10 mmol) andtriethylamine (2ml, 15 mmol) in dry dioxan (50 ml) was heated at 100° C.for 4 hours. After cooling to ambient temperature, the reaction mixturewas filtered and the filtrate concentrated under reduced pressure. Theresidual oil was chromatographed on silica gel (eluted initially withdichloromethane: methanol, 98:2, changed to dichloromethane; methanol,95:5) to give the title compound as an oil (2.7 g, 70%). IR: ν_(max)(film) 3200, 2995, 1690, 1640, 1600, 1570, 1240, 1165, 1030 cm⁻¹. ¹ HNMR: δ_(H) [(CD₃)₂ SO+D₂ O] 1.33 (6H,t,J=7Hz, (OCH₂ CH₃)₂), 3.76 (2H,m,--CH₂ OCH₂ OP, 3.86 (2H,d,J=8Hz, OCH₂ P), 4.0-4.1 ( 6H,m, N--OCH₂ +(OCH₂CH₃)₂), 8.15 (1H,s,H-2) Found: C,37.10; H,5.52; N,14.27%; m/z382.0807(M⁺). C₁₂ H₂₀ N₄ O₆ PCl requires: C,37.66; H,5.27; N,14.64%; m/z382.0809 (M⁺).

b) 6-Chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]purine

A solution of4-chloro-6-[2-(diethoxyphosphorylmethoxy)ethoxyamino]-5-formamidopyrimidine(2.4 g, 6.3 mmol) in diethoxymethyl acetate (10 ml) was heated at 120°C. for 45 minutes. After cooling to ambient temperature, the solvent wasremoved under reduced pressure. The residue was dissolved in methanol(15 ml) and 0.880 ammonia (1 ml). After 15 minutes, the reaction mixturewas evaporated to dryness and the residue chromatographed on silica gel(eluted with dichloromethane: methanol, 98:2) to give the title compoundas an oil (2.15 g, 94%). IR: ν_(max) (film) 3100, 3070, 2995, 1600,1570, 1440, 1395, 1335, 1250, 1220, 1170, 1030, 930, 640 cm⁻¹. ¹ H NMR:δ_(H) [(CD₃)₂ SO] 1.23 (6H,t,J=7Hz, (OCH₂ CH₃)₂), 3.88 (2H, d, J=8Hz,OCH₂ P), 3.89 (2H,m, CH₂ OCH₂ P), 4.0-4.1 (4H,m, (OCH₂ CH₃)₂), 4.61(2H,m, N--OCH₂), 8.82 (1H,s,H-8), 8.97 (1H,s,H-2). Found: C,38.38;H,5.10; N,15.14%. C₁₂ H₁₈ N₄ O₅ PCl. 0.5H₂ O requires: C,38.56; H,5.12;N,14.99%. Found: m/z 364.0703. C₁₂ H₁₈ N₄ O₅ PCl requires: m/z 364.0701.

Description 3 Intermediates for Examples 3, 4 (Method B) and 5

a)4-Chloro-6-2-(diethoxyphosphorylmethoxy)ethoxyamino]-2,5-diformamidopyrimidine

A mixture of 4,6-dichloro-2,5-diformamidopyrimidine (2.35 g, 10 mmol),diethyl 2-(aminooxy)ethoxymethylphosphonate (2.27 g, 10 mmol), anddiisopropylethylamine (3.48 ml, 20 mmol) in diglyme (40 ml) was heatedat 100° C. for 3 hours. After cooling to ambient temperature, thesolvent was evaporated under reduced pressure and the residue obtainedchromatographed on silica gel (eluted with dichloromethane methanol97.3) to give the title compound as a yellow foam (2.8 g, 65%). ¹ H NMR:δ_(H) [(CD₃)₂ SO] 1.26 (6H, t, J=7Hz, (OCH₂ CH₃)₂), 3.76 (2H, m CH₂ OCH₂P), 3.88 (2H, d, J=8Hz, OCH₂ P), 3.95-4.10 (6H, m, N--OCH₂ +(O--CH₂CH₃)₂), 8.14 (1H, s, NHCHO), 9.16+9.41 (combined 1H, D₂ O exchangeableNHCHO), 9.24 (1H, br.s, NHCHO), 10.85 (2H, br.s, D₂ O exchangeable,NHOCH₂ +NHCHO).

b) 6-Chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]-2-formamidopurine

A solution of 4-chloro-6-[2-(diethoxyphosphorylmethoxy)ethoxyamino]-2,5-diformamidopyrimidine (2.8 g, 6.6 mmol) indiethoxymethyl acetate (20 ml) was heated at 120° C. for 2 hours. Aftercooling to ambient temperature, excess solvent was removed under reducedpressure, the residue dissolved in methanol (20 ml) and 0.880 ammonia(6.5 ml) and the solution stirred at ambient temperature for 1 hour. Thesolvent was evaporated to leave an oil which was chromatographed onsilica gel (eluted with dichloromethane: methanol, 98.2) to give thetitle compound as a pale yellow oil (2.4 g, 90%). IR: ν_(max) (film)3480, 3120, 2995, 1710, 1610, 1580, 1510, 1440, 1390, 1330, 1240, 1050,1030, 970, 920, 780 cm⁻¹ ; ¹ H NMR δ_(H) [(CD₃)₂ SO] 1.23 (6H, t, J=7Hz, (OCH₂ CH₃)₂), 3.87 (2H, d, J=8 Hz, OCH ₂ P), 3.87 (2H, m, N-OCH₂CH₂), 4.03 (4H, m, (OCH₂ CH₃)₂), 4.56 (2H, m, N-OCH₂ CH₂), 8.75 (1H, s,H-8), 9.38 (1H, br.s, CHO), 11.30 (1H, br.s, D₂ O exchangeable NHCHO).Found: C, 38.19; H, 4.79; N, 16.74%. C13H₁₉ N5O₆ PCl requires: C, 38.29;H, 4.70; N, 17.17%. Found: m/z 408.0827 (MH⁺). C13H₂₀ N₅ O₆ PClrequires: m/z 408.0840 (MH⁺).

Description 4 Intermediates (V) for Examples 6 (Method B), 7, 8, 9(Method A) and 10

a) Diethyl [2-benzyloxy-1-(benzyloxymethyl)ethoxy]methylphoshonate

Dry HCl gas was bubbled through an ice-cooled solution of1,3-dibenzyloxypropan-2-ol (25 g, 0.092 mol) and paraformaldehyde (2.75g, 0.092 mol) in dry dichloromethane (100 ml) for 1 hour. The resultingsolution was dried (MgSO₄) and evaporated to dryness to leave an oil.Triethyl phosphite (15.7 ml, 0.092 mol) was added and the resultingmixture stirred and heated at 140° C. for 16 hours. The liquid obtainedwas dissolved in ethyl acetate and washed with sodium bicarbonatesolution. The organic phase was washed with brine, dried (MgSO₄) andevaporated to give a mobile oil, which was chromotographed in silica(hexane/ethyl acetate 50:50 as eluant) to give the title compound as acolourless, mobile oil (19.7 g, 51). IR: ν_(max) (film) 3055, 3020,2900, 2860, 1490, 1470, 1450, 1390, 1305, 1255, 1095, 1050, 1030, 970,820, 770, 735, 700 cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃) 1.3 (6H, t, J=7 Hz,P-(OCH₂ CH₃)₂), 3.6 (4H, d, 2×CH₂ OCH₂ Ph),3.8-4.05 (1H, m, CH), 4.05(2H, d, J=9 Hz, OCH₂ P), 4.0-4.3 (4H, m, P-(OCH₂ CH₃)₂), 4.55 (4H, s,2×OCH₂ Ph), 7.38 (10H, s, 2×Ph). C₂₂ H₃₁ PO₆ requires: C, 62.54; H,7.42. Found: C, 62.75, H, 7.61. m/z: C₂₂ H₃₁ PO₆ requires 422.1858;observed (M⁺) 422.1864.

b) Diethyl [2-hydroxy-1-(hydroxymethyl)ethoxy]methylphosphonate andDiethyl [2-benzyloxy-1-(hydroxymethyl)ethoxy]methylphosphonate

To a solution of diethyl[2-benzyloxy-1-(benzyloxymethyl)ethoxy]methylphosphoate (15.5 g, 36.7mmol) in ethanol (200 ml), containing a trace of methanolic HCl (1 ml),was added 10% Pd-C (1.5 g). The mixture was treated with hydrogen atatmospheric temperature and pressure for a total of 6 days. Filtrationand evaporation of the solvent gave an oil which was chromotagraphed onsilica (eluant dichloromethane/methanol 95:5, then 92:8) after whichwere obtained the monobenzyl ether (3.65 g, 30%) and the diol (2.73 g,30%).

Data for diol. IR: ν_(max) (film) 3400, 2980, 2940, 2920, 1650, 1480,1445, 1390, 1370, 1295, 1230, 1165, 1120, 1020, 980, 880, 820, 780 cm⁻¹.¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.25-3.6(5H, m, CH₂ CHCH₂), 3.94 (2H, d, J=8.2 Hz, OCH₂ P), 4.0-4.2 (4H, m,(OCH₂ CH₃)₂), 4.56 (2H, br.s, D₂ O exchangeable, 2×OH). m/z: C₈ H₂₀ O₆ Prequires: 243.0998; observed 243.0986 (M+H⁺). Data for monobenzyl ether.IR: ν_(max) (film) 3395, 2980, 2900, 2860, 1475, 1450, 1390, 1365, 1240,1160, 1090, 1050, 1020, 970, 810, 775, 730, 695 cm⁻¹. ¹ H NMR: δ_(H)[(CD₃)₂ SO] 1.21, 1.20 (6H, 2×t, J=7 Hz, 7 Hz, (O-CH₂ CH₃)₂) 3.5-3.7(5H, m, CH₂ CHCH₂ -), 3.95 (2H, d, d, J=8.8 Hz, OCH₂ P), 4.0 (4H, m,(OCH₂ CH₃)₂), 4.49 (2H, s, OCH₂ Ph), 4.68 (1H, t, D₂ O exchangeable OH),7.2-7.5 (5H, m, Ph). C₁₅ H₂₅ O₆ P. 0.4 H₂ O requires: C, 53.06; H, 7.67.Found: C, 53.11; H, 7.94. m/z: C₁₅ H₂₆ O₆ P requires 333.1467, observed:333.1472 (M+H⁺).

c) Diethyl2-benzyloxy-1-(N-phthalimidooxymethyl)ethoxy]methylphosphonate

Diethyl azodicarboxylate (1.82 ml, 11.6 mmol) was added to a solution ofdiethyl [2-benzyloxy-1-(hydroxymethyl) ethoxy]methylphosphonate (3.5 g,10.5 mmol), N-hydroxyphthalimide (1.72 g, 10.5 mmol) andtriphenylphosphine (3.04 g, 11.6 mmol) in dry THF (60 ml). The reactionmixture was left at ambient temperature for 5 days, then evaporated todryness. The residue obtained was dissolved in diethyl ether and left at5° C. for 16 hours. The precipitated crystals of triphenylphosphineoxide were filtered off and the ether filtrate evaporated to dryness toleave an oil. After chromatography on silica gel (eluant hexane/ethylacetate 50:50, then 30:70, then 20:80) the title compound was obtainedas a colourless oil (4.1 g, 80%). IR: ν_(max) (film) 3060, 3020, 2980,2930, 2920, 1790, 1740, 1465, 1450, 1370, 1250, 1190, 1160, 1130, 1100,1080, 1050, 1030, 970, 870, 820, 780, 740, 700 cm⁻¹. ¹ H NMR: δ_(H)(CDCl₃) 1.30 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.7 (2H, d, J=5H_(z), CHCH₂OCH₂ Ph), 4.0-4.25 (7H, m, (OCH₂ CH₃)₂, OCH₂ Ph, CH₂ CHCH₂), 4.36 (2H,m, CH₂ ON), 4.56 (2H, s, OCH₂ Ph), 7.30-7.35 (5H, br.s, Ph), 7.74-7.9(4H, m, phthalimide aromatic H). m/z: C₂₃ H₂₈ NO₈ P requires 477.1553;observed: 477.1548 (M⁺).

d) Diethyl (1-aminooxymethyl-2-bezyloxyethoxy)methylphosphonate

To a solution of diethyl[2-benzyloxy-1-(N-phthalimidooxymethyl)ethoxy]methylphosphonate, (4 g,8.4 mmol) in dry dichloromethane (40 ml), was added methylhydrazine(0.54 ml, 10.1 mmol) at ambient temperature and the mixture stirred for2 hours. The solution was filtered and the filtrate evaporated todryness. The residue was dissolved in ether and left at 5° C. overnight.The deposited solid was filtered off, the filtrate evaporated and theresidue chromatographed on silica (eluant dichoromethane/methanol 98:2)to give the title compound as a colourless oil (2.7 g, 92%). ¹ H NMR:δ_(H) (CDCl₃) 1.32, 1.33 (6H, 2xt, P-(OCH₂ CH₃)₂), 3.5-3.65 (2H, m,CHONH₂), 3.78 (2H, d, CH₂ OCH₂ Ph), 3.95-4.1 (3H, m, OCH₂ P+CH₂ CHCH₂),4.1-4.3 (4H, m, P-(OCH₂ CH₃)₂), 4.53 (2H, s, OCH₂ Ph), 5.0-6.0 (2H,br.s, D₂ O exchangeable, NH₂), 7.27-7.35 (5H, m, aromatic H). m/z: C₁₅H₂₆ NO₆ P requires 347.1498; observed 347.1516 (M⁺).

DESCRIPTION 5 Intermediates for Examples 6 (Method B), 7 and 8

a)6-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxyamino-4-chloro-5-formidopyrimidine

A mixture of 4,6-dichloro-5-formamidopyrimidine (390 mg, 2 mmol),diethyl (1-aminooxymethyl-2-benzyloxyethoxy)methylphosphonate (700 mg, 2mmol) and triethylamine (0.41 ml, 3 mmol) in dry dioxan (5 ml) washeated at 100° C. for 2 hours. The reaction mixture was cooled, filteredand the filtrate evaporated to leave a yellow oil which waschromatographed on silica (dichloromethane/methanol 98:2 as eluant) togive the title compound as a yellow viscous oil (750 mg, 74%), ¹ H NMR:δ_(H) [(CD₃)₂ SO+D₂ O] 1.30 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.55-3.8 (2H,m, CH₂ OCH₂ Ph), 3.9-4.2 (9H, m, (OCH₂ CH₃)₂ +OCH₂ P+NHOCH₂ CH), 4.6(2H, s, OCH₂ Ph), 7.42 (m, 5H, aromatic Ph), 8.24 (1H, s, H-2). Found:C, 47.66; H,5.69; N, 10.51%. C₂₆ H₂₈ N₄ O₇ PCl requires: C, 47.76; H,5.61; N,11.14%, m/z: C₂₀ H₂₈ N₄ O₇ PCl requires: 502.1384; observed:502.1340 (M⁺).

b) 9-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy-6-chloropurine

A solution of6-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxyamino]-4-chloro-5-formamidopyrimidine(750 mg, 1.49 mmol) in diethoxymethyl acetate (2 ml) was stirred andheated at 120° C. for 2 hours. After cooling to ambient temperature, theexcess solvent was evaporated. The residue was dissolved in methanol (10ml) and 0.880 ammonia (1 ml) and left at ambient temperature for 10minutes. The solvent was removed under reduced pressure and the residuechromatographed on silica (dichloromethane/methanol, 98:2 as eluant) togive the title compound as a yellow oil (590 mg, 80%). IR: ν_(max)(film) 3060, 2990, 2905, 1590, 1565, 1435, 1330, 1250, 1220, 1165, 1100,1050, 1030, 970, 930, 850 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.21 (6H, t,J=7 Hz, (OCH₂ CH₃)₂), 3.6-3.75 (2H, m, CH₂ OCH₂ Ph) 3.8-4.1 (7H, m,(OCH₂ CH₃)₂ +OCH₂ P+CH₂ CHCH₂), 4.53 (2H, s, OCH₂ Ph), 4.5-4.7 (2H, m,N-OCH₂), 7.32 (5H, br.s, Ph), 8.82 (1H, s), 9.02 (1H, s) Found: C,49.71; H, 5.68; N, 10 94%. C₂₀ H₂₆ N₄ O₆ PCl requires: C, 49.54; H,5.40; N, 11.56%. m/z: C₂₀ H₂₆ N₄ O₆ PCl requires 484.1279; observed 4841249 (M⁺).

c) 9-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine

A solution of9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloropurine (570mg, 1.18 mmol) in ethanolic ammonia (10 ml) was heated in a sealedvessel at 110° C. for 2 hours. After cooling to ambient temperature, thesolvent was evaporated and the residue obtained chromatographed onsilica (dichloromethane/methanol 95:5 as eluant) to give the titlecompound (350 mg, 64%). IR: ν_(max) (film) 3320, 3200, 2980, 2900, 640,1595, 1470, 1450, 1410, 1390, 1370, 1330, 1290, 1240, 1160, 1090, 1050,1020, 970, 820, 790, 730, 700, cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.22(6H, t, J=7 Hz, OCH₂ CH₃)₂), 3.6-3.75 (2H, m, CH₂ OCH₂ Ph), 3.95-4.2(7H, m, (OCH₂ CH₃)₂ +CH₂ CHCH₂ +OCH₂ P), 4.4-4.65 (2H, m, N-OCH₂), 4.52(2H, s, OCH₂ Ph), 7.25-7.4 (5H, m, aromatic), 7.39 (2H, br.s, D₂ Oexchangeable NH₂), 8.15(1H, s), 8.41 (1H, s). Found: C, 49.81; H, 5.75;N, 14.07%; C₂₀ H₂₈ N₅ O₆ P. H₂ O requires: C, 49.68; H, 6.25; N, 14.48%,m/z: C₂₀ H₂₈ N₅ O₅ P requires 465.1777; observed: 467.1757.

DESCRIPTION 6 Intermediates for Examples 9 (Method A) and 10

a)6-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxyamino]-4-chloro-2,5-diformamidopyrimidine

A mixture of 4,6-dichloro-2-5-diformamidopyrimidine (650 mg, 2.74 mmol),diethyl (1-aminooxymethyl-2-benzyloxyethoxy)methylphosphonate (950 mg,2.74 mmol) and diisopropylethylamine (0.95 ml, 5.5 mmol) in diglyme (12ml) was heated at 100° C. for 21/2 hours. The mixture was cooled and thesolvent evaporated to leave an oily residue which was chromatographed onsilica (dichloromethane/methanol 97:3 as eluant) to give the titlecompound as a yellow foam (800 mg, 54%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.21(6H, 2×t, P-(OCH₂ CH₃)₂), 3.5-3.7 (2H, m, CH₂ OCH₂ Ph), 3.75-4.2 (9H, m,(OCH₂ CH₃)₂ +OCH₂ CH+OCH₂ P), 4.51 (2H, s, OCH₂ Ph), 7.25-7.5 (5H, m,Ph), 8.15 (1 H, s, CHO), 9.26 (1H, br.s, CHO), 9.44-10.88 (3H, 2×br.s,D₂ O exchangeable NH+2×NHCHO). Found: C, 46.32; H, 5.61; N, 11.94% C₂₁H₂₉ N5O₈ PCl requires: C, 46.20; H, 5.35; N, 12.83%. m/z: 545 (M⁺).

b)9-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloro-2-formamidopurine

A solution of6-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxyamino]-4-chloro-2,5-diformamidopyrimidine(760 mg, 1.4 mmol) in diethoxymethyl acetate (2 ml) was stirred andheated in an oil bath at 120° C. for 2 hours. After cooling to ambienttemperature, the solvent was evaporated and the residue dissolved inmethanol (10 ml) and 0.880 ammonia (1 ml). After 15 minutes at ambienttemperature the solution was evaporated to dryness. The residue obtainedwas chromatographed on silica gel (dichloromethane/methanol, 98:2 aseluant) to give the title compound as a colourless oil (550 mg, 75%).IR: ν_(max) (film), 3400, 3220, 3110, 2980, 2900, 1710, 1610, 1580,1510, 1475, 1440, 1385, 1330, 1240, 1160, 1140, 1095, 1050, 1020, 980,920, 820, 780, 740, 700 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.20, 1.21 (6H,2×t, P-(OCH₂ CH₃)₂), 3.6-3.75 (2 H, m, CHCH₂), 3.8-4.15 (7H, m, CH₂CHCH₂, OCH₂ P, P-(OCH₂ CH₃)₂), 4.52 (2H, s, OCH₂ Ph), 4.45-4.65 (2H, m,N-OCH₂), 7.25-7.40 (5H, m, Ph), 8.81 (1H, s, H-8), 9.37 (1H, br.s,NHCHO), 11.31 (1H, br.s, D₂ O exchangeable NHCHO). Found: C, 48.11; H, 537; N, 12.50%. C₂₁ H₂₇ N₅ O₇ PCl requires: C, 47.77; H, 5.16; N, 13.27%.m/z: C₂₁ H₂₈ N₅ O₇ PCl requires 528.1415; observed 528.1363 (M +H⁺).

c) 9-[3-Benzyloxy-2-(diethoxyphosphorylmethoxy)propoxylquanine

A solution of9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloro-2-formamidopurine(520 mg, 1 mmol) in 80% aqueous formic acid (6 ml) was stirred andheated at 80° C. for 5 hours. After cooling and evaporation to dryness,the residue was dissolved in methanol (5 ml) and 0.880 ammonia (1 ml).After 15 minutes at ambient temperature, the solvent was evaporated andthe residue obtained chromatographed on silica gel(dichloromethane/methanol 90:10 as eluant) to give the title compound asa white solid (330 mg, 70%), mp. 166°-169° (acetone). IR: ν_(max) (KBr)3326, 3167, 2983, 2907, 2868, 2745, 1694, 1648, 1600, 1586, 1540, 1475,1454, 1391, 1328, 1251, 1163, 1100, 1051, 1027, 970, 823, 787, 739, 693,624, cm⁻¹. UV: λ_(max) (EtOH) 255 nm (14,100), ¹ H NMR: δ_(H) [(CD₃)₂SO], 1.22 (6H, 2 ×t, J=7 Hz, P-(OCH₂ CH₃)₂), 3.5-3.8 (2H, m, CH₂ OCH₂Ph), 3.85-4.25 (7H, m, OCH₂ P, P-(OCH₂ CH₃)₂, CH₂ CHCH₂), 4.3-4.5 (2H,m, N-OCH₂), 4.50 (2H, s, OCH₂ Ph), 6.58 (2H, br.s, D₂ O exchangeableNH₂), 7.25-7.5 (5H, m, Ph), 7.95 (1H, s, H-8), 10.69 (1H, br.s, D₂ Oexchangeable NH). Found: C, 49.35; H, 5.85; N, 14.60%. C₂₀ H₂₈ N₅ O₇ P.O.25H₂ O requires: C, 49.43; H, 5.91; N, 14.41%. m/z: (FAB, matrixthioglycerol)₄₈₂ (M+H⁺).

DESCRIPTION 7 Intermediates (V) for Examples 6 (Methods A and C). 7, 8,9 (Method B), 10 15 and 16

a) Diethyl 2-acetoxy-1-(hydroxymethyl)ethoxy]methylphosphonate

To a solution of diethyl [2-hydroxy-1-(hydroxymethyl)ethoxy]methylphosphonate (6.1 g, 25.2 mmol) in dry THF (75 ml) were addedp-toluenesulphonic acid (250 mg) and trimethyl orthoacetate (4.45 ml, 35mmol). The reaction mixture was stirred at ambient temperature for 16hours after which water (1 ml) plus 2MHCl (5 drops) were added. Afterstirring for a further 30 minutes, the solution was evaporated todryness and the residue chromatographed on silica gel (eluantdichloromethane: methanol 97:3) to give the title compound (5.1 g, 71%)as colourless mobile oil. IR: ν_(max) (film) 3400, 2980, 2920, 2910,1740, 1440, 1390, 1370, 1245, 1160, 1120, 1050, 1030, 970, 820, 780cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃) 1.36 (6H, t, J=7 Hz, P(OCH₂ CH₃)₂), 2.10(3H,s, COCH₃), 3.65-4.50 (12H,m, P(OCH₂ CH₃)₂, OCH₂ P, OCH₂ CHCH₂ OH).Found: C,41.79; H,7.66%. C₁₀ H₂₁ O₇ P requires: C,42.25; H,7.66%. m/z:(Isobutane C.I) 285 (MH⁺, 100%).

b) Diethyl [2-acetoxy-1-(N-phthalmidooxymethyl)ethoxy]methylphosphonate

Diethyl azodicarboxylate (1.89 ml, 12 mmol) was added to a solution ofdiethyl [2-acetoxy-1-(hydroxymethyl)ethoxy]methylphosphonate (3.1 g,10.9 mmol), triphenylphosphine (3.15 g, 12 mmol) andN-hydroxyphthalimide (1.78 g, 10.7 mmol) in dry THF (60 ml). Thereaction mixture was stirred at ambient temperature under an atmosphereof nitrogen for 16 hours, and then evaporated to dryness. The residueobtained was dissolved in diethyl ether and left at 4° C. for 16 hours.The crystals of triphenylphosphine oxide were removed by filtration andthe filtrate evaporated to dryness. The residue was chromatographed onsilica gel (eluant hexane:acetone 70:30) to give the title compound as apale yellow oil (3.6 g, 76). IR: ν_(max) (film) 3500, 3460, 2980, 2900,1780, 1735, 1465, 1440, 1370, 1240, 1185, 1160, 1030, 1020, 970, 875,820, 780, 700 cm⁻¹. ¹ H NMR: δ_(H) (CDCl₃) 1.33 (6H,2×t, J=7 Hz, (OCH₂CH₃)₂, 2.09 (3H,s, COCH₃), 3.9-4.5 (11H,m, (OCH₂ CH₃)₂, +OCH₂ P,+OCH₂CHCH₂), 7.75-7.87 (4H,m, aromatic H). Found: C,50.14; H,5.69%; N, 3.15%.C₁₈ H₂₄ NO₉ P requires: C,50.35; H,5.63; N, 3.26%. m/z: observed430.1275; C₁₈ H₂₅ NO₉ P (MH⁺) requires 430.1267.

c) Diethyl [2-acetoxy-1-(aminooxymethyl)ethoxyl]methylphosphonate

To a solution of diethyl[2-acetoxy-1-(N-phthalimidooxymethyl)ethoxy]methylphosphonate (1.0 g.2.33 m mol), in dry dichloromethane (20 ml) was added methylhydrazine(0.125 ml, 2.35 mmol) and the solution stirred at ambient temperaturefor 10 minutes. The reaction mixture was filtered, and the filtrateevaporated to dryness. The residue obtained was chromatographed onsilica gel (eluant dichloromethane methanol 98:2) to give the titlecompound as a colourless oil (0.59 g, 86%). IR: ν_(max) (film) 3460,3320, 3240, 3170, 2995, 2910, 1740, 1595, 1445, 1390, 1370, 1240, 1165,1115, 1050, 1025, 975, 820, 780 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23(6H,t, J=7 Hz, (OCH₂ CH₃)₂), 2.02 (3H,s, COCH₃), 3.5-3.65 (2H,m, NOCH₂),3.8-4.25 (9H,m, OCH₂ P) (OCH₂ CH₃)₂ +CHCH₂), 6.13 (2H br. s, D₂ Oexchangeable NH₂). Found: C,39.52; H,7.20; N,4.46%; C₁₀ H₂₂ NO₇ Prequires: C,40.13; H,7.41; N, 4.68%. m/z: observed 299.1117; C₁₀ H₂₂ NO₇P (M⁺) requires 299.1134.

DESCRIPTION intermediates for Examples 6 (Method A), 7 and 8

a)6-[[3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]amino]-4-chloro-5-formamidopyrimidine

A mixture of 4,6-dichloro-5-formamidopyrimidine (0.77 g, 4.0 mmol),diethyl [2-acetoxy-1-(aminooxymethyl)ethoxy]methylphosphonate (1.2 g,4.0 mmol) and triethylamine (0.82 ml, 6 mmol) in dry dioxan (20 ml) washeated at 100° C. for 2 hours. The reaction mixture was cooled,filtered, and the filtrate evaporated to leave an oil which waschromatographed on silica gel (eluant dichloromethane:methanol 97:3) andgave the title compound as a yellow oil (1.08 g, 60%). ¹ H NMR: δ_(H)[(CD₃)₂ SO] 1.23 (6H,t, J=7 Hz, (OCH₂ CH₃)₂), 2.03 (3H,s, COCH₃),3.85-4.35 (11H,m, (OCH₂ CH₃)₂ +OCH₂ P+OCH₂ CHCH₂ O). m/z: observed454.1020; C₁₅ H₂₄ N₄ O₈ PCl (M⁺) requires 454.0996.

b) 9-3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloropurine

A solution of6-[[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]amino]-4-chloro-5-formamidopyrimidine(0.80 g, 1.76 mmol) in diethoxymethyl acetate (5 ml) was heated at 120°C. for 2 hours. After cooling to ambient temperature, the excess solventwas evaporated under reduced pressure. The residue obtained wasdissolved in methanol (5 ml) and .880 ammonia (1 ml) and left at ambienttemperature for 10 minutes. The solvent was removed under reducedpressure and the residue chromatographed on silica gel (eluantdichloromethane:methanol 98:2) to give the title compound as a yellowoil (0.68 g, 88). IR: ν_(max) (film) 3080, 3050, 2990, 2900, 1740, 1640,1590, 1565, 1435, 1390, 1370, 1330, 1240, 1160, 1120, 1040, 1025, 970,930, 850, 820, 780 cm⁻¹. ¹ M NMR: δ_(H) [(CD₃)₂ SO] 1.23 (6H,t, J=7 Hz,(OCH₂ CH₃)₂). 2.04 (3H,s, COCH₃), 3.95-4.15 (7H,m, (OCH₂ P+(OCH₂ CH₃)₂+CH), 4.20 (1H,dd, J=5.22 Hz, J=12.1 Hz, CH_(B) OAc), 4.36 (1H,dd,J=3.85 Hz, J=12.1 Hz, CH_(A) OAc), 4.55 (1H,dd, J=6.05 Hz, J=11.55 Hz,N-OCH_(B)), 4.70 (1H,dd,J=3.58 Hz, J=11.55 Hz, N-OCH_(A)), 8.83 (1H,s),9.04(1H,s). Found: C,41.38; H,5.13; N,12.39%; C₁₅ H₂₂ N₄ O₇ PClrequires: C,41.24; H,5.08; N,12.83%. m/z: (FAB, thioglycerol matrix) 437(MH⁺).

DESCRIPTION 9 Intermediates for Example 16

a)6-[[3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]amino]-4-chloro-2,5-diformamidopyrimidine

A mixture of 4,6-dichloro-2,5-diformamidopyrimidine (0.475 mg, 2 mmol),diethyl [2-acetoxy-1-(aminooxymethyl)ethoxy)methylphosphonate (0.60 mg,2 mmol) and triethylamine (0.54 ml, 4 mmol) in dry dioxan (20 ml) washeated at 120° C. for 5 hours. The reaction mixture was cooled toambient temperature, filtered, and the filtrate evaporated to leave ayellow oil. The oil was chromatographed on silica gel (eluantdichloromethane:methanol 97:3) to give the title compound as a paleyellow solid (0.81 g, 80%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23 (2×3H, 2×t,J=7 Hz, (OCH₂ CH₃)₂), 2.03 (3H,s, COCH₃), 3.80-4.35 (11H,m, CH₂ CHCH₂+(OCH₂ CH₃)₂ +OCH₂ P), 8.16 (1H,br.s, sharpens to s. on D₂ O, CHO), 9.26(1H,d,J=9.9 Hz. collapses to s. on D₂ O. CHO), 9.45 (1H,br.s, D₂ Oexchangeable, NH), 10.8-10.95 (2H,m, D₂ O exchangeable 2×NH). Found:C,37.36; H,5.37; N,13.43%; C₁₆ H₂₅ N₅ O₉ PCl.H₂ O requires: C,37.18;H,5.27; N,13.55%. m/z: observed 497.1082; C₁₆ H₂₅ N₅ O₉ PCl(M⁺) requires497.1078.

b)9-[3-Acetoxy-2-(diethoxyohosphorylmethoxy)propoxy]-6-chloro-2-formamidopurine

A solution of6-[[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]amino]-4-chloro-2,5-formamidopyrimidine(0.30 g, 0.66 mmol) in diethoxymethyl acetate (5 ml) was heated at 120°C. for 3 hours. After cooling to ambient temperature, excess solvent wasremoved under reduced pressure. The residue obtained was dissolved in

methanol (2 ml) and 0.880 ammonia (0.5 ml) and left at ambienttemperature for 15 minutes. The solvent was removed under reducedpressure and the residue obtained chromatographed on silica gel (eluantdichloromethane:methanol 97:3) to give the title compound as acolourless gum (0.20 g, 63%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.22 (2×3H,2×t,J=7 Hz, (OCH₂ CH₃)₂), 2.04 (3H,s, COCH₃), 3.75-4.20 (7H,m, (OCH₂CH₃)₂ +OCH₂ P, CH), 4.21 (1H,dd, J=5.22 Hz, J=12.10 Hz, CH_(B) OAc) 4.38(1H,dd, J=3.85 Hz, J=12.10 Hz, CHOAc), 4.50 (1H,dd, J=6.05 Hz, J=11.55Hz, NOCH_(B)), 4.63 (1H,dd, J=3.57 Hz, J=11.55 Hz,NOCH_(A)), 8.82 (1H,s,H-8), 9.38 (1H,d, J=9.6 Hz, collapses to s. on D₂ O, CHO), 11.32(1H,d,J=9.6 Hz, D₂ O exchangeable NH).

DESCRIPTION 10 Intermediates for Examples 6 (Method A), 7 and 8

a) 9-Benzyloxy-6-chloropurine

A mixture of 4,6-dichloro-5-formamidopyrimidine (58.6 g; 0.31 mmol),benzyloxyamine (37.5 g; 0.31 mmol), triethylamine (110 ml) and dioxan(400 ml) was stirred at 100° C. for 4 hours. The reaction was cooled,filtered and evaporated under reduced pressure. The residue waspartitioned between ethyl acetate (750 ml), saturated aqueous potassiumbicarbonate (400 ml) and brine (200 ml). The organic phase was separatedand the aqueous phase washed with ethyl acetate (300 ml). The combinedorganic phases were washed with water (200 ml), brine (200 ml), dried(MgSO₄), and evaporated under reduced pressure.

The residue was dissolved in anhydrous N,N-dimethylformamide (100 ml),triethyl orthoformate (200 ml), and 12N hydrochloric acid (5 ml). After4 hours at 25° C. the solvent was removed under reduced pressure. Theresidue was partitioned between chloroform (750 ml) and saturatedaqueous potassium bicarbonate (500 ml). The resulting suspension wasfiltered and the phases separated. The organic phase was washed withsaturated potassium bicarbonate (200 ml), water (200 ml), dried (MgSO₄)and evaporated under reduced pressure. The residue was purified bycolumn chromatography on silica gel eluting with chloroform-methanol(100:1), affording the title compound (38.4 g, 48%). IR: ν_(max) (KBr)3350, 1587, 1565, 1438 cm⁻¹ ; ¹ H NMR: δ_(H) (CDCl₃) 5.40(2H, s, CH₂Ar), 7.35(5H, s, Ar), 7.75(1H, s, H-8), 9.85(1H, s, H-2). Found: C,55.11; H, 3.73; N, 21.27%. C₁₂ H₉ N₄ OCl requires: C, 55.28; H, 3.49, N,21.50%.

b) 9-Benzyloxyadenine

A solution of 9-benzyloxy-6-chloropurine (38.4 g; 0.147 mmol) in ethanol(300 ml) saturated with ammonia was heated at 100° C. in an autoclavefor 16 hours. After cooling the suspension was evaporated to dryness andthe residue partitioned between chloroform (750 ml) and water (500 ml).The separated aqueous phase was washed with chloroform (200 ml). Thecombined organic phases were washed with water, dried (MgSO₄) andevaporated, affording an orange solid homogeneous on t.l.c. (31.1 g,87%). IR: ν_(max) (KBr) 3372, 3300, 3187, 3038, 1660, 1637, 1600, 1581cm⁻¹ ; ¹ H NMR: δ_(H) [(CD₃)₂ SO] 5.3(2H, s, CH₂ Ar), 6.8(2H, br.s, D₂ Oexchangeable, NH₂), 7.3(6H, s, H-8, Ar), 7.7(1H, s, H-2). Found: C,59.39; H, 4.60; N, 29.07%; m/e 241 0949. C₁₂ H₁₁ N₅ O requires: C,59.73; H, 4.60; N, 29.03%; m/z 241.0964.

c) 9-Benzyloxy-6-phthalimidopurine

Phthaloyl dichloride (13.35 g; 92.2 mmol) was added to a cooled solutionof 9-benzyloxyadenine (14.9 g, 61.5 mmol), 4-dimethylaminopyridine (1.5g, 12.3 mmol) and triethylamine (25.7 ml, 184.4 mmol) in tetrahydrofuran(200 ml). After 1 hour at room temperature the solvent was removed underreduced pressure and the residue partitioned between chloroform (500 ml)and saturated potassium bicarbonate (300 ml). The organic phase wasseparated, washed with water (200 ml), brine (200 ml), dried (MgSO₄) andevaporated to dryness. Column chromatography on silica gel eluting withchloroformmethanol (100:1) afforded the title compound (11.20 g, 49%).IR: ν_(max) (KBr) 3070, 1800, 1740, 1730, 1605, 1585, 1450 and 1410 cm⁻¹; ¹ H NMR δ_(H) [(CD₃)₂ SO] 5.70(2H, s, CH₂ Ph), 7.55(5H, s, CH₂ Ph),8.20(4H, s, Ar), 8.95(1H, s, H-2), 9.25(1H, s, H-8). Found: C, 64.71; H,3.78; N, 18.85%; m/z 371.1025. C₂₀ H₁₃ N₅ O₃ requires: C, 64.68; H,3.54; N, 18.86%; m/e 371.1018.

d) 9-Hydroxy-6-phthalimidopurine

A mixture of 9-benzyloxy-6-phthalimidopurine (11.0 g, 29.5 mmol), 10%palladium on charcoal (2.2 g), ethanol (300 ml) and tetrahydrofuran (500ml) was stirred at 25° C. for 1 hour under an atmosphere of hydrogen.The suspension was then filtered and the catalyst washed with ethanol.The filtrate was evaporated under reduced pressure and the resultingsolid triturated with ether. The solid was collected and then dried toafford the title compound (6.93 g; 83%). IR: ν_(max) (KBr). 2607, 1794,1735, 1603, 1582, 1467, 1401 cm⁻¹ ; ¹ H NMR δ_(H) [(CD₃)₂ SO] 8.15(4H,s, Ar), 8.95(1H, s, H-2), 9.15(1H, s, H-8), 12.80(1H, br.s, D₂ Oexchangeable, OH). Found: C, 55.34; H, 2.58; N, 24.56%. C₁₃ H₇ N₅ O₃requires: C, 55.51; H, 2.51; N, 24.91%.

e)9-[3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurine

Diethyl azodicarboxylate (0.4 ml, 2.6 mmol) was added to a mixture of9-hydroxy-6-N-phthalimidopurine (0.61 g, 2.17 mmol), diethyl[2-acetoxy-1-(hydroxymethyl)ethoxy]methylphosphonate (0.61 g, 2.17 mmol)triphenylphosphine (0.68 g, 2.6 mmol) in dry THF (20 ml) at 0° C. A paleyellow solution was obtained within a few minutes and the reactionmixture was then stirred at ambient temperature for 2 hours. The solventwas evaporated under reduced pressure and the residue obtainedchromatographed on silica gel using hexane: acetone 3:1 as eluant,before changing to acetone: hexane 3:1 to give the title compound as acolourless oil (0.95 g, 80%). IR: ν_(max) (film) 3100, 3060, 2980, 2910,1790, 1740, 1730, 1600, 1580, 1465, 1445, 1405, 1370, 1330, 1240, 1160,1135, 1020, 980, 880, 790, 775 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23(2×3H, 2×t, J=7 Hz, (OCH₂ CH₃)₂). 2.05 (3H,s, COCH₃), 3.95-4.45 (9H,m,OCH₂ CH₃)₂ +OCH₂ P, CHCH₂ OAc), 4.55-4.80 (2H,m, N-OCH₂), 8.0-8.20(4H,m, aromatic H), 9.04 (1H,s) 9.11(1H,s). m/z: (FAB, thioglycerolmatrix) 548 (MH⁺, 100).

b) 9-[3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine

To a solution of9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurine(0.91 g, 1.66 mmol) in dry dichloromethane (5 ml) was addedmethylhydrazine (0.09 ml, 1.1 eq) and the solution stirred at ambienttemperature for 15 minutes. The reaction mixture was filtered, the whitesolid washed with dichloromethane and the combined filtrates evaporatedto dryness. The residue obtained was chromatographed on silica gel(dichloromethane:methanol 95:5 as eluant) to give the title compound asa colourless oil (0.58 g, 83). IR: ν_(max) (film) 3330, 3190, 2990,2900, 1740, 1660, 1640, 1595, 1470, 1410, 1370, 1330, 1295, 1240, 1050,1020, 975, 820, 790 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23 (2×3H, 2×t,J=7 Hz, (OCH₂ CH₃)₂), 2.03 (3H,s, COCH₃), 4.0-4.15 (7H,m, (OCH₂ CH₃)₂+OCH+OCH ₂ P), 4.15-4.3-7 (2H, 2xdd, J=12.10, 5.2, 4.1 Hz, C₂₀ Ac), 4.44(1H,dd, J=11.3 H_(z),J=6.0 Hz,NOCH_(B)), 4.55 (1H, dd, J=11.27 Hz,J=3.85 Hz, NOCHA), 7.38 (2H,br.s,D₂ O exchangeable NH₂), 8.15 (1H,s),8.43(1H,s). Found: C,42.01; H,5.86; N,16.14%; C₁₅ H₂₄ N₅ O₇ P. 0.5H₂ O.requires: C,42.25; H,5.91; N,16.42%. m/z: observed 417.1412; C₁₅ H₂₄ N₅O₇ P requires: 417.1412.

DESCRIPTION 11 Intermediates for Examples 9 (Method B), 10, 15 and 20

a) 6-Benzyloxyamino-4-chloro-2,5-diformamidopyrimidine

A mixture of 4,6-dichloro-2,5-diformamido-pyrimidine (1.9 g, 8.09 mmol),benzyloxyamine (1 g, 8.13 mmol), triethylamine (2 ml) and dioxan (20 ml)was stirred at 100° C. for 1 hour. The cooled reaction mixture wasfiltered and the precipitate collected and washed with dioxan (2×5 ml).The filtrate and washings were combined and evaporated to a syrup.Column chromatography on silica gel (eluted with chloroform-ethanol,30:1) afforded the title compound (1.2 g, 46%). IR: ν_(max) (KBr) 3242,1694, 1588, 1472 cm⁻¹ ; ¹ H NMR δ_(H) [(CD₃)₂ SO], 4.89 (2H, s, OCH₂Ph), 7.4 (5H, m, Ph), 8.15 (1H, s, CHO), 9.18, 9.42 (1H, 2×br.s, D₂ Oexchangeable, NH), 9.25 (1H, br.s, CHO), 10.91 (2H, br.s, D₂ Oexchangeable, 2×NH). m/z (FAB+ve ion, thioglycerol) MH⁺ 322.

b) 9-Benzyloxy-6-chloro-2-formamidopurine

6-Benzyloxyamino-4-chloro-2,5-diformamidopyrimidine (1.2 g, 3.73 mmol)and diethoxymethyl acetate (20 ml) was stirred at 120° C. for 2.5 hours,cooled and evaporated under reduced pressure. A solution of the residuein methanol (20 ml) and 0.880 ammonia (2 ml) was stirred at 20° C. for 1hour, the solvent removed under reduced pressure and the residueco-evaporated with methanol. Column chromatography on silica gel (elutedwith chloroform-ethanol, 100:1) afforded the title compound (700 mg,62%). IR: ν_(max) (KBr) 3119, 1702, 1611, 1577, 1505, 1440 cm⁻¹ ; ¹ HNMR: δ_(H) [(CD₃)₂ SO], 5.44 (2H, s, CH₂ Ph), 7.45 (5H, m, Ph), 8.54(1H, s, H-8), 9.34 (1H, s, CHO), 11.30 (1H, br.s, D₂ O exchangeable,NH). Found: C, 49.99; H, 3.37; N, 22.43%, m/z 303.0523. C₁₃ H₁₀ N₅ O₂Cl+0.5 H₂ O requires: C, 49.92; H, 3.55; N, 22.40%, m/z 303.0520.

c) 2-Amino-9-benzyloxy-6-methoxypurine

A mixture of 9-benzyloxy-6-chloro-2-formamidopurine (440 mg, 1.60 mmol),1.2M sodium methoxide in methanol (5.3 ml) and methanol (10 ml) washeated at reflux temperature for 1 hour and then cooled. Acetic acid (4ml) was added and the solution evaporated to dryness. The residue wassuspended in water and extracted with chloroform (2×25 ml). The combinedchloroform extracts were washed with brine, dried (magnesium sulphate)and evaporated under reduced pressure. Column chromatography on silicagel (eluted with chloroform-methanol, 100:1) afforded the title compound(331 mg, 76%). IR: ν_(max) (KBr) 3480, 3310, 1625, 1585, 1505, 1485,1460, 1400 cm⁻¹ ; ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.96 (3H, s, CH₃), 5.31(2H, s, CH₂ Ph), 6.64 (2H, br.s, D₂ O exchangeable, NH₂), 7.42 (5H, s,Ph), 7.75 (1H, s, H-8). Found: C, 57.18, H, 4.84; N, 25.85%). m/z271.1075. C₁₃ H₁₃ N₅ O₂ requires: C, 57.56, H, 4.83, N, 25.82%; m/z271.1069.

d) 9-Benzyloxy-2-(bis-t-butoxycarbonyl)amino]-6-methoxypurine

A solution of 2-amino-9-benzyloxy-6-methoxypurine (0.47 g; 1.73 mmol),di-t-butyldicarbonate (0.57 g; 2.60 mmol) and4-N,N-dimethylaminopyridine (100 mg, 0.173 mmol) in tetrahydrofuran washeated at reflux for 45 minutes. Additional di-t-butyldicarbonate (0.20g) was then added and the solution refluxed for 30 minutes. The reactionwas then cooled and the solvent removed under reduced pressure. Theresidue was purified by column chormatography on silica gel eluting withchloroform-methanol mixtures, affording the title compound (740 mg;91%). IR: ν_(max) (KBr) 3110, 2990, 1760, 1600, 1485, 1460, 1400 cm⁻¹ ;¹ H NMR: δ_(H) (CDCl₃) 1.50(18H, s, 6×CH₃), 4.15(3H, S, CH₃), 5.45(2H,s, CH₂), 7.35(5H, s, Ar), 7.65(1H, s, H-8).

e) 2-[(Bis-t-butoxycarbonyl)amino}-9-hydroxy-6-methoxypurine

A mixture of 9-benzyloxy-2-[(bis-t-butoxycarbonyl)amino]-6-methoxypurine(990 mg; 2.10 mmol), 10% palladium on charcoal (100 mg), ethanol (25 ml)and dioxan (25 ml) was stirred at 20° C. under an atmosphere of hydrogenfor 45 minutes. The suspension was then filtered and the filtrateevaporated under reduced pressure. The resulting white solid was driedto yield the title compound (760 mg; 95). IR: ν_(max) (KBr) 2990, 2420,1760, 1740, 1730, 1710, 1605, 1480 cm⁻¹ ; ¹ H NMR δ_(H) [(CD₃)₂ SO]1.40(18H, s, 6×CH₃), 4.05(3H, s, OCH₃), 8.05(1H, s, H-8), 11.8(1H, br.s,D₂ O exchangeable, OH) Found: C, 50.27; H, 6.12; N, 17.70%. C₁₆ H₂₃ N₅O₆ +0.2EtOH requires: C, 50.42; H, 6.23; N, 17.66%.

f)9-3-Acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]2-[bis-(t-butoxycarbonyl)amino-6-methoxypurine

Diethyl azodicarboxylate (0.49 ml, 3.15 mmol) was added to a mixture of2-[bis-(t-butoxycarbonyl)amino]-9-hydroxy-6-methoxypurine (1 g, 2.6mmol) triphenylphosphine (0.82 g, 3.15 mmol) and diethyl[2-acetoxy-1-(hydroxymethyl)ethoxy]methylphosphonate (0.74 g, 2.6 mmol)in dry THF (20 ml) at 0° C. The reaction mixture was stirred at ambienttemperature for 3 hours, the solvent removed under reduced pressure andthe residue obtained chromatographed on silica gel (haxane:acetone 3:1as eluant, then hexane:acetone 1:1 and finally hexane:acetone 1:3) togive the title compound as a colourless oil (1 g, 59%). IR: ν_(max)(film) 2980, 2940, 1790, 1740, 1595, 1490, 1425, 1370, 1280, 1250, 1165,1120, 1100, 1050, 970, 850, 790 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.22(2×3H, 2×t, J=7 Hz, (OCH₂ CH₃)₂), 1.41 (18H,s, 2×C(CH₃)₃), 2.02 (3H,s,COCH₃), 3.75-4.40 (9H,m, (OCH.sub. 2 CH₃)₂ +OCH₂ P+CHCH₂ OAc), 4.08(3H,s,OCH₃), 4.48 (1H, dd, J=6.05 Hz, J=11.27 Hz, NOCH_(B)), 4.58(1H,dd,J=3.3 Hz, J=11.27 Hz, NOCHA), 8.76 (1H,s,H-8). m/z: (FAB,thioglycerol matrix) 648 (MH⁺, 9%).

DESCRIPTION 12

a) (S)-1-Benzyloxy-3-(t-butyldiphenylsilyloxy)propan-2-ol

To a solution of (R)-3-benzyloxypropane-1,2-diol (prepared from(S)-4-benzylymethyl-2,2-dimethyl-1,3-dioxolane (commercially availablefrom FLUKA) by acid hydrolysis using 80% aqueous acetic acid), (8.3 g,47.2 mmol) in dry THF (70 ml) was added imidazole (6.42 g, 94.4 mmol).After 2 minutes, t-butyldiphenylsilyl chloride (12.28 ml, 47.2 mmol) wasadded. A white precipitate formed immediately. After 2 hours, thereaction mixture was filtered and the filtrate evaporated. The residuewas partitioned between ethyl acetate and water, the organic layerseparated, washed with saturated brine, dried (MgSO₄), filtered andevaporated to give a colourless oil. Chromatography of the oil on silicagel (dichloromethane as eluant) afforded the title compound as acolourless oil (17.72 g, 90%).

IR ν_(max) (film) 3580, 3070, 2940, 2860, 1595, 1475, 1455, 1430, 1395,1365, 1345, 1310, 1270, 1265, 1240, 1110, 1030, 1010, 1000, 960, 940,828, 810, 745, 710 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 0.97 (9H, s, t-Bu),3.4-3.85 (5H, m, CH₂ CHCH₂), 4.50 (2H, s, OCH₂ Ph), 4.84 (1H, d, J=5.3Hz, D₂ O exchangeable OH), 7.25-7.70 (15H, m, aromatic H). Found C,74.27, H, 7.71%. C₂₆ H₃₂ O₃ Si requires: C, 74.24; H, 7.67%. m/z: (NH₃C.I.) 438 (MNH₄ +, 8%). [α]_(D) ²⁵ (CHCl₃) -1.7° (C=0.94).

b) Diethyl(S)-[2-benzyloxy-1-(t-butyldiphenylsilyloxymethyl)ethoxy]methylphosphonate

Dry HCl gas was bubbled through a solution of(S)-1-benzyloxy-3-(t-bttyldiphenylsilyloxy)propan-2-ol (4.206 g, 0.01mmol) and paraformaldehyde (0.3 g, 0.01 mmol) in dry dichloromethane (20ml) containing anhydrous calcium chloride (7.5 g), for 1 hour. Anyremaining traces of water were removed by drying over magnesiumsulphate. The solution was filtered and evaporated to dryness to give acolourless oil. Triethyl phosphite (1.71 ml, 0.01 mmol) was added andthe solution was heated at 140° C. for 5 hours, then allowed to cool toambient temperature. The reaction product was chromatographed on silicagel (dichloromethane: methanol, 99:1 as eluant) to give the titlecompound as a colourless oil (2.25 g, 40%).

IR: ν_(max) (film) 3060, 2960, 2930, 2900, 2850, 1475, 1455, 1430, 1390,1365, 1260, 1115, 1055, 1030, 960, 820, 805, 740, 710 cm⁻¹. 1H NMR:δ_(H) (CDCl₃) 1.03 (9H, s, t-Bu), 1.28 (2×3H, 2×t, J=7 Hz, (OCH₂ CH₃)₂),3.5-3.85 (5H, m, CH₂ CHCH₂), 3.96 (2H, d, J=8.9 Hz, OCH₂ P), 4.05-4.25(4H, m, (OCH₂ CH₃)₂), 4.52 (2H, s, OCH₂ Ph), 7.25-7.75 (15H, m, aromaticH). Found: C, 65.43; H, 7.70%. C₃₁ H₄₃ O₆ PSi requires: C, 65.24; H,7.59%. m/z: (NH₃ C.I.) 588 (MNH₄ ⁺, 35%), 571 (MH⁺, 100%). [α]D²⁵(CHCl₃) -6.9° (C=1.06).

DESCRIPTION 13 [(R)-ISOMERS] Diethyl(S)-[1-(t-butyldiphenylsilyloxymethyl)-2-hydroxyethoxy]methylphosphonate

To a solution of diethyl(S)-[2-benzyloxy-1-(t-butyldiphenylsilyloxymethyl)ethoxy]methylphosphonate(1.90 g, 3.3 mmol) in ethanol (20 ml) containing a trace of methanol/HCl(10 drops), was added 10%Pd-C (1.2 g). The mixture was shaken under anatmosphere of hydrogen at ambient temperature and atmospheric pressureuntil hydrogen uptake was complete and t.l.c. (SiO₂, CH₂ Cl₂ -MeOH,95:5) indicated no starting material remained. The reaction mixture wasfiltered, the filtrate evaporated and the residue obtainedchromatographed on silica gel (dichloromethanemethanol, 95:5 as eluant)to give the title compound as a colourless oil (1.35 g, 84%).

IR: ν_(max) (film) 3420, 3080, 3060, 2945, 2870, 1595, 1475, 1430, 1395,1365, 1245, 1115, 1060, 1030, 975, 825, 745, 715 cm⁻¹. ¹ H NMR: δ_(H)[(CD₃)₂ SO]1.0 (9H, s, t-Bu), 1.20 (2×3H, 2×t, J=7.1 Hz, (OCH₂ CH₃)₂),3.45-3.60 (3H, m, CH₂ CH), 3.65-3.80 (2H, m, CH₂), 3.90-4.15 (6H, m,OCH₂ P,(OCH₂ CH₃)₂), 4.65 (1H, t, J=5.2 Hz, D₂ O exchangeable OH),7.30-7.75 (1OH, m, aromatic H). Found: C, 60.11; H, 7.87%.C₂₄ H₃₇ O₆ PSirequires: C, 59.97; H, 7.76%. m/z: (NH₃ C.I.) 498 (MNH₄ ⁺, 15%), 481(MH⁺, 100%). [α]D²⁵ (CHCl₃) -19.0° (C=0.4).

DESCRIPTION 14 [(S)-ISOMERS] Diethyl(R)-[2-benzyloxy-1-(hydroxymethyl)ethoxy]-methylphosphonate

A suspension of diethyl(S)-[2-benzyloxy-1-(t-butyldiphenylsilyloxymethyl)ethoxy]methylphosphonate(3.09 g, 5.4 mmol) in trifluoroacetic acid/water 2:1 (12 ml) was stirredat ambient temperature for 1 hour. The solution obtained was extractedwith hexane (2×10 ml) and the aqueous containing phase evaporated todryness. The residue obtained was treated with ethanolic ammonia (5 ml)for 5 minutes at ambient temperature, the solvent evaporated and theresidue chromatographed on silica gel using dichloromethane/methanol95:5 as eluant. The title compound was obtained as a colourless oil(1.35 g, 75%). IR: ν_(max) (film) 3380, 3060, 3030, 2980, 2910, 2860,1495, 1480, 1450, 1390, 1370, 1250, 1160, 1100, 1075, 975, 910, 890,820, 780, 740, 700 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO]1.21, 1.22 (2×3H,2×t, J=7 Hz, (OCH₂ CH₃)₂, 3.4-3.65 (5H, m, CH₂, CH,CH₂), 3.94 (2H, d,J=8.5 Hz, OCH₂ P), 3.97-4.1 (4H, m, (OCH₂ CH₃)₂), 4.5 (2H, s, CH₂ Ph),4.67 (1H, t, J=5.5 Hz, D₂ O exchangeable OH), 7.20-7.40 (5H, m, Ph).Found: C, 53.20; H, 7.72%. C₁₅ H₂₅ O₆ P 0.33 H₂ O requires: C, 53.25; H,7.64%. m/z: observed 332.1383. C₁₅ H₂₅ O₆ P requires 332.1387. [α]D²⁵(CHCl₃) +16.7° (c=1.06).

EXAMPLES

Compounds of formula (I) prepared as hereinbefore described [R₃ /R₄ whenjoined--see formula (I)].

    __________________________________________________________________________    Ex. No.                                                                             R.sub.1                                                                            R.sub.2                                                                           R.sub.3                                                                            R.sub.4                                                   __________________________________________________________________________    1     NH.sub.2                                                                           H   H    P(O)(OEt).sub.2                                           2     NH.sub.2                                                                           H   H    P(O)(OH).sub.2                                            3     OH   NH.sub.2                                                                          H    P(O)(OEt).sub.2                                           4     OH   NH.sub.2                                                                          H    P(O)(OH).sub.2                                            5     OH   NH.sub.2                                                                          H    P(O)(OH)(OEt)                                             6     NH.sub.2                                                                           H   CH.sub.2 OH                                                                        P(O)(OEt).sub.2                                           7     NH.sub.2                                                                           H   CH.sub.2 OH                                                                        P(O)(OH).sub.2                                            8     NH.sub.2                                                                           H   CH.sub.2 OH                                                                        P(O)(OH)(OEt)                                             9     OH   NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OEt).sub.2                                           10    OH   NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OH).sub.2                                            11    NH.sub.2                                                                           NH.sub.2                                                                          H    P(O)(OEt).sub.2                                           12    NH.sub.2                                                                           NH.sub.2                                                                          H    P(O)(OH).sub.2                                            13    Cl   NH.sub.2                                                                          H    P(O)(OH).sub.2                                            14    OCH.sub.3                                                                          NH.sub.2                                                                          H    P(O)(OH).sub.2                                            15    OH   NH.sub.2                                                                            joined (R.sub.6 = H)                                         16    NH.sub.2                                                                           NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OH).sub.2                                            17    OH   NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OH).sub.2                                                                          (R)-isomer                                      18    OH   NH.sub.2                                                                            joined (R.sub.6 = H)                                                                       (S)-isomer                                      19    NH.sub.2                                                                           H   CH.sub.2 OH                                                                        P(O)(OH).sub.2                                                                          (R)-isomer                                      20    OH   NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OH).sub.2                                                                          (S)-isomer                                      21    OH   NH.sub.2                                                                            joined (R.sub.6 = H)                                                                       (R)-isomer                                      22    NH.sub.2                                                                           H   CH.sub.2 OH                                                                        P(O)(OH).sub.2                                                                          (S)-isomer                                      23    NH.sub.2                                                                           NH.sub.2                                                                          CH.sub.2 OH                                                                        P(O)(OH).sub.2                                                                          (R)/(S)-isomers                                 24    NH.sub.2                                                                           H     joined (R.sub.6 = H)                                                                       (R)/(S)-isomers                                 25    NH.sub.2                                                                           NH.sub.2                                                                            joined (R.sub.6 = H)                                                                       (R)/(S)-isomers                                 __________________________________________________________________________

EXAMPLE 1 9-[2-(Diethoxyphosphorylmethoxy)ethoxy]adenine

A solution of 6-chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]purine(1.45 g, 4 mmol) in ethanol (20 ml) saturated with ammonia gas washeated in a sealed steel vessel at 100° C. for 2 hours. The reactionmixture was cooled to ambient temperature, evaporated to dryness and theresidual oil chromatographed on silica gel (eluted with dichloromethane:methanol, 90:10) to give recovered starting material (0.25 g) and thetitle compound (0.94 g, 82%--based on used starting material),crystallised from acetone-ether, m.p. 74°-76°. UV: λ_(max) (EtOH) 260 nm(ε 13,500); IR: ν_(max) (KBr) 3308, 3146, 1669, 1646, 1600, 1538, 1466,1416, 1320, 1301, 1232, 1206, 1164, 1129. 1066, 1053, 1039, 1009, 978cm⁻¹ ; ¹ H NMR: δ_(H) [(CD₃)₂ SO]1.24 (6H,t,J=7 Hz, (OCH₂ CH₃)₂),3.83-3.89 (2H,m,CH₂ OCH₂ P), 3.91 (2H,d,J=8 Hz, OCH₂ P), 4.0-4.15 (4H,dq, J=7 Hz, 7 Hz, (OCH₂ CH₃)₂), 4.48-4.51 (2H,m,N-OCH₂), 7.37(2H,br.s,D₂ O exchangeable NH₂), 8.14 (1H,s,H-8), 8.34 (1H,s,H-2).Found: C,41.74; H: 5.49; N: 20.06%; m/z 345.1202. C₁₂ H₂₀ N₅ O₅ Prequires: C,41.74; H, 5.83; N, 20.28%; m/z 345.1212.

EXAMPLE 2 9-[2-(Phosphonomethoxy)ethoxy]adenine

To a solution of 9-[2-(diethoxyphosphorylmethoxy)ethoxy]adenine (0.63 g,1.83 mmol) in dry dichloromethane (10 ml) was added trimethylsilylbromide (1.45 ml, 11 mmol) and the solution stirred at ambienttemperature for 2 hours. The solvent was removed under reduced pressureand the residue dissolved in methanol (10 ml). The solvent wasevaporated and the residue again dissolved in methanol and solventevaporated to give a solid residue. Crystallisation from methanol:acetone gave the title compound which was recrystallised from methanol:water (0.24 g, 45%), m.p. 241°-244° C. UV: λ_(max) (EtOH) 259 nm (ε13,500); IR: ν_(max) (KBr) 3428, 3312, 3084, 1685, 1644, 1612, 1484,1462, 1455, 1336, 1294, 1278, 1255, 1215, 1195, 1104, 1060, 1046, 1033,955, 934, 892 cm⁻¹. ¹ H NMR: δH [(CD₃)₂ SO]3.64 (2H,d,J=8.2 Hz, OCH₂ P),3.82 (2H,m,N-OCH₂ CH₂ O), 4.50 (2H,m, N-OCH₂ CH₂), 7.42 (2H, br.s, D₂ Oexchangeable NH₂), 8.15 (1H,s,H-8), 8.38 (1H,s,H-2) 10.0-4.5 (2H,broad,D₂ O exchangeable PO(OH )₂). Found: C,32.84; H,4.41; N: 23.74%. C₈ H₁₂N₅ O₅ P. H₂ O requires: C,33.01; H,4.19; N:24.06%.

EXAMPLE 3 9-[2-(Diethoxyphosphorylmethoxy)ethoxy]quanine

A solution of6-chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]-2-formamidopurine (2.1g, 5.1 mmol) in 80% aqueous formic acid (15 ml) was stirred and heatedat 80° C. for 1.5 hours. After cooling to ambient temperature, thereaction mixture was evaporated to dryness under reduced pressure, theresidue dissolved in methanol (20 ml) and .880 ammonia (2 ml) and leftat ambient temperature for 1 hour. The solvent was evaporated and theresidue obtained chromatographed on silica gel (eluted withdichloromethane: methanol, 90:10) to give the title compound (1.5 g,80%), which was crystallised from ethanol (0.8 g, 43%) m.p. 176°-178°.UV: ν_(max) (H₂ O) 253 (ε 13,300)nm; λ_(sh) (H₂ O) 275 (ε 9050)nm; IR:ν_(max) (KBr) 3334, 3156, 1693, 1646, 1601, 1590, 1242, 1163, 1054,1025, 970 cm⁻¹ ; ¹ H NMR: δ_(H) [(CD₃)₂ SO]1.24 (6H,t,J=7 Hz, (OCH₂CH₃)₂), 3.80 (2H,m, N-OCH₂ CH₂ O), 3.90 (2H,d,J=8 Hz, OCH₂ P), 4.06(4H,dq,J=7 Hz,7 Hz, (OCH₂ CH₃)₂), 4.39 (2H,m, N-OCH₂ CH₂), 6.60(2H,br.s, D₂ O exchangeable, NH₂), 7.89 (1H,s,H-8), 10.67 (1H,br.s, D₂ Oexchangeable N-1(H)). Found: C,39.45; H,5.47; N,19.05%. C₁₂ H₂₀ N₅ O₆ Prequires: C,39.89; H,5.58; N,19.39%.

EXAMPLE 4 9-[2-(Phosphonomethoxy)ethoxy]quanine Method A

To a solution of 9-[2-(diethoxyphosphorylmethoxy)ethoxy]quanine (100 mg,0.28 mmol) in dry dimethylformamide (2 ml) was added trimethylsilylbromide (0.22 ml, 1.66 mmol). The reaction mixture was stirred for 16hours at ambient temperature, evaporated to dryness and the residueobtained dissolved in methanol (5 ml). After 5 minutes at ambienttemperature, the methanol was removed by evaporation. The residue wasagain treated with methanol as indicated above. The solid obtained wasinsoluble in water and methanol. This solid was suspended in water and0.880 ammonia added until the solution pH reached 9-10. The basicsolution was added to Dowex 50W-X8 resin (prewashed with 0.1M sodiumhydroxide and then water until the washings were neutral) and the resinwashed with water. Fractions containing UV absorbing material werecombined and acidified with 2M HCl to precipitate a solid. Theprecipitate was collected and triturated with methanol to give the titlecompound as a white solid (50 mg, 60%), m.p. 275°- 278° C. UV: ν_(max)(EtOH) 254nm (ε 10800); λ_(sh) (EtOH) 278 nm (ε 6400); IR: ν_(max) (KBr)3378, 3161, 1705, 1649, 1602, 1459, 1382, cm⁻¹ ; ¹ H NMR: δ_(H) [(CD₃)₂SO] 3.60 (2H,d,J=8.25 Hz, OCH₂ P), 3.77 (2H,m,-CH₂ OCH₂ P), 4.37 (2H,m,N-OCH₂ CH₂), 6.63(2H,br.s, D₂ O exchangeable NH₂), 7.92 (1H,s,H-8);10.67 (1H,br.s,D₂ O exchangeable NH), 2.6-5.5 (2H,broad, phosphonic acidH). Found: C,30.79; H,4.24; N,21.96%. C₈ H₁₂ N₅ O₆ P. 0.5H₂ O requires:C,30.59; H,4.17; N,22.30%.

Method B

To a solution of6-chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]-2-formamidopurine (0.25g, 0.61 mmol) in methanol (5 ml), was added sodium methoxide solution(30 wt % in methanol; 0.5 ml, 2.62 mmol). The mixture was stirred andheated at 50° C. for 4 hours. After cooling to ambient temperature, 80%aqueous acetic acid (0.5 ml) was added to give a solution of pH₆. Thesolution was evaporated to dryness and the residue partitioned betweendichloromethane (10 ml) and aqueous sodium bicarbonate solution (10 ml).The aqueous phase was washed with dichloromethane (2×10 ml), thecombined organic extracts dried (magnesium sulphate), and evaporated todryness under reduced pressure. The residue obtained was chromatographedon silica gel (eluant dichloromethane: methanol 90:10) to give a2-amino-6-methoxypurine derivative (0.20 g) which was used without anyfurther purification. The chromatographed material (0.20 g) wasdissolved in dichloromethane (2 ml), trimethylsilyl bromide (0.7 ml, 5.3mmol) added, and the solution left at ambient temperature for 2 days.The solvent was evaporated under reduced pressure and the residueobtained co-evaporated with methanol (×2) to leave a white solid whichcrystallised from hot water giving the title compound as a white solid(0.105 g, 56%) m.p. 275°-278° C., identical in all respects with thecompound obtained in Method A.

EXAMPLE 5 9-[2-[Ethoxy(hydroxy)phosphorylmethoxy)ethoxy]quanine

A solution of 9-[2-(diethoxyphosphorylmethoxy)ethoxy]quanine (100 mg,0.28 mmol) in 10% (w/w) sodium hydroxide solution (3 ml) was heatedunder reflux for 4 hours. After cooling to ambient temperature, thesolution was neutralised with 12M HCl and evaporated to dryness to givea yellow solid. The solid was purified by chromatography on reversephase silica eluting initially with water, then methanol-water (5:95)and finally methanol-water (10:90). UV absorbing fractions werecollected and evaporated to dryness to leave the title compound as awhite solid, m.p. 244°-246° (25 mg, 27%).¹ H NMR: 67 H [(CD₃)₂ SO+C₅ D₅D] 1.18 (3H, t, J=7 Hz, OCH₂ CH₃), 3.74 (2H, d, J=8.5 Hz, OCH₂ P),3.80-3.85 (2H, m, CH₂ OCH₂ P), 3.98 (2H, m, P-OCH₂ CH₃), 4.40-4.45 (2H,m, N-OCH₂), 7.07 (2H, br.s, D₂ O exchangeable, NH₂), 8.01 (1H, s, H-8),11.40 (1H, br.s, D₂ O exchangeable, NH), 4.1-4.7 (br.s, P-OH+water insolvent). Found: C, 35.34; H, 4.78; N, 20.40%. C₁₀ H₁₆ N₅ O₆ P. O.5H₂ Orequires: C, 35.09; H, 5.00; N, 20.46%.

EXAMPLE 6 9-[2-(Diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenineMethod A

A solution of 9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine(0.58 g, 1.39 mmol) in ethanol (10 ml) and 2MHCl (1.5 ml, 3.0 mmol) washeated under reflux for 16 hours. The solution was cooled to ambienttemperature and evaporated to dryness. The residue obtained wasdissolved in ethanol and 0.880 ammonia solution added to give a finalsolution of pH 8.5. The solvent was removed under reduced pressure andthe residue obtained chromatographed on silica gel (eluantdichloromethane:methanol 95:5, then 92:8, and finally 90:10) to give thetitle compound as a pale yellow oil (0.42 g, 80%). ¹ H NMR: δ_(H)[(CD₃)₂ SO] 1.23 (6H,t,J=7 Hz, (OCH₂ CH₃)₂), 3.57 (2H,m, CH₂ OH), 3.79(1H,m,CH), 3.95-4.15 (6H,m, OCH₂ P, (OCH₂ CH₃)₂), 4.38 (1H,dd.J=6.6 Hz,J=11.27 Hz, NOCH_(B)), 4.54 (1H,dd,J=3.03 Hz, J=11.27 Hz, NOCH_(A)),4.88 (1H,t, D₂ O exchangeable OH), 7.38 (2H,br.s,D₂ O exchangeable NH₂),8.15 (1H,s), 8.43 (1H,s). Found: C,38.72; H,6.06; N,17.50%; Cl₃ H₂₂ N₅O₆ P.1.5H₂ O requires: C,38.80; H,6.01; N,17.41%. m/z: observed:375.1306; C₁₃ H₂₂ N₅ O₆ P(M⁺) requires: 375.1308.

Method B

To a solution of9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine (300 mg,0.65 mmol) in 80% aqueous acetic acid (10 ml) was added 10% Pd-C (30 mg)and the mixture shaken under a hydrogen atmosphere at ambienttemperature and pressure for a total of 8 hours. The solution wasfiltered and the filtrate evaporated. The residue obtained waschromatographed on silica (dichloromethane/methanol 95:5, then 90:10 aseluants) to give the title compound, (100 mg, 40%).

Method C

A solution of9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloropurine (0.63g, 1.44 mmol) in ethanolic ammonia (12 ml) was heated in a sealed vesselat 100° C. for 5 hours. After cooling to ambient temperature, thesolvent was removed under reduced pressure to leave a brown solid. Thesolid was chromatographed on silica gel (dichloromethane:methanol 90:10as eluant) and separated into9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine (0.29 g) and9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenine. The acetatederivative was hydrolysed as in Method A, and the product of thehydrolysis combined with the initially chromatographed alcohol to givethe title compound as a pale yellow oil (0.30 g, 55%).

EXAMPLE 7 9-[3-Hydroxy-2-(phosphonomethoxy)propoxy]adenine and EXAMPLE 89-[2-[ethoxy(hydroxy)phosphorylmethoxy]-3-hydroxypropoxy]adenine

To a solution of9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenine (90 mg, 0.24mmol) in dry dichloromethane (2.5 ml) and dry DMF (0.5 ml), was addedbromotrimethylsilane (0.19 ml, 1.44 mmol). After 2 hours stirring atambient temperature, the solvent was evaporated and the residue obtaineddissolved in methanol. The solution was evaporated to dryness again andthe residue again dissolved in methanol and evaporated. The residue waschromatographed on reverse phase silica [water (2 column volumes), then5% methanol:water (3 column volumes), then 10% methanol: water (3 columnvolumes) as eluants] to give the diacid (17 mg, 22%), mp 197°-203°, andthe monoacid-monoester (30 mg, 36%) mp 90°-95°, after lyophilisation.

Data for diacid, (Example 7), IR: ν_(max) (KBr disc) 3550-2100 (broad),1704, 1608, 1467, 1418, 1340, 1304, 1229, 1207, 1163, 1125, 1079, 925,889, 778, 757, 730 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.54 (2H, m, CHCH₂OH), 3.75 (3H, m, OCH₂ P +CH₂ CHCH₂), 4.3-4.56 (2H, m, N-OCH₂), 7.39(2H, br.s, D₂ O exchangeable NH₂), 8.15 (1H, s), 8.48 (1H, s), 3.45 (3H,br.s, D₂ O exchangeable PO(OH)₂ +CH₂ OH) UV: λ_(max) (EtOH) 259 nm.Found: C, 31.02; H, 4.50; N, 19.85%. C₉ H₁₄ N₅ O₅ P. 1.5H₂ O requires:C, 31.22; H, 4.94; N, 20.23%. m/z: (FAB. thioglycerol matrix) 320 (MH⁺).

Data for monoester, (Example 8). IR: ν_(max) (KBr) 3600-2200 (broad),1692, 1647, 1602, 1472, 1413, 1333, 1296, 1166, 1128, 1047, 953, 882,795, 781, 729. UV: νmax (EtOH) 259 nm. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.9(3H, t, J=7 Hz, P-(OCH₂ CH₃), 3.54 (2H, m, CHCH₂ OH), 3.74 (1H, m, CHCH₂OH) 3.83 (2H, d, J=8.8 Hz, OCH₂ P), 3.96 (2H, m, P-OCH₂ CH₃),4.3-4.55(2H, m, N-OCH₂), 7.39 (2H, br.s, D₂ O exchangeable NH₂), 8.15(1H, s), 8.46 (1H, s), 3.4 (2H, br.s, D₂ O exchangeable P-OH+CH₂ OH).Found: C, 37.10; H, 5.29; N, 19.59%. C₁₁ H₁₈ N₅ O₆ P. 0.5 l H₂ Orequires: C, 37.08; H, 5.37; N, 19.65%. m/z: (FAB: thioglycerol matrix)348 (MH⁺).

EXAMPLE 9 9-2-(Diethoxyphosphorylmethoxy)-3-hydroxypropoxy]quanineMethod A

A mixture of 9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]quanine(300 mg, 0.6 mmol) and 5% Pd-C (30 mg) in 80% aqueous acetic acid (10ml) was shaken under a hydrogen atmosphere at ambient temperature andpressure until hydrogen uptake had ceased--a total of 16 hours. Thehydrogenation mixture was filtered and the filtrate evaporated. Theresidue was chromatographed on silica gel (dichloromethane/methanol85:15 as eluant) to give the title compound as a white solid (84 mg,30%) mp. 129°-133°. IR:ν_(max) (KBr) 3335, 3150, 2984, 2933, 1693, 1648,1602, 1476, 1382, 1244, 1164, 1116, 1053, 1026, 972 cm⁻¹. ¹ H NMR: δ_(H)[(CD₃)₂ SO] 1.23 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.45-3.65 (2H, m, CH₂OH), 3.7-3.85 (1H, m, CH₂ CHCH₂), 3.9-4.2 (6H, m, (OCH₂ CH₃)₂ +OCH₂ P),4.2-4.5 (2H, m, N-OCH₂ CH), 4.84 (1H, t, J=5.5 Hz, D₂ O exchangeableOH), 6.6 (2H, s, D₂ O exchangeable NH₂), 7.96 (1H, s, H-8), 10.69 (1H,br.s, D₂ O exchangeable NH). Found; C, 39.15; H, 5.50; N, 17.14%. C₁₃H₂₂ N₅ O₇ P. 0.5H₂ O requires C, 39.00; H, 5.79; N, 17.49%. m/z:(positive ion FAB) 392 (M⁺ H⁺).

Method B

A solution of9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-2-[bis-t-butoxycarbonyl)amino]-6-methoxypurine(0.28 g) 0.43 mmol) in ethanol (4 ml) and 2MHCl (1 ml) was heated underreflux for 5 hours. The solution was cooled and evaporated to dryness.The residue obtained was chromatographed on silica gel (pre-absorbedonto silica gel in ethanol; dichloromethane:methanol 80:20 as eluant) toafford 9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]quanine as apale yellow solid (0.08 g, 47%).

EXAMPLE 10 9-[3-Hydroxy-2-(phosphonomethoxy)propoxy]quanine

To a solution of9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]quanine (60 mg, 0.15mmol) in dry dimethylformamide (1 ml) was added bromotrimethylsilane(0.5 ml, 3.75 mmol). The solution was kept at ambient temperature for 3hours after which the solution was evaporated to dryness under reducedpressure. The residue obtained was dissolved in methanol and thesolution evaporated to dryness again to leave an off-white solid. Thissolid was triturated with methanol to give the title compound as a whitesolid, m.p.>300°, (34 mg, 66%). IR ν_(max) (KBr). 3385, 3159, 1715,1645. 1598, 1384, 1157. 1109, 1060, 930, 772 cm⁻¹. ¹ H NMR: δ_(H)[(CD₃)₂ SO], 3.52 (2H, m, CH₂ OH), 3.60-3.80 (3H, m, OCH₂ P+CH₂ CH) 4.25(1H, dd, J=11 Hz, J=7.1 Hz, N-OCH_(B)), 4.40 (1 H, dd, J=11 Hz, J=3.3 HzN-OCH_(A)), 6.61 (2H, br.s, D₂ O exchangeable NH₂), 8.00 (1H, s, H-8),10.65 (1H, br.s, D₂ O exchangeable NH) 2.6-4.1 (3H, broad, D₂ Oexchangeable OH, PO(OH)₂). Found: C, 31.59; H, 4.11; N, 20.42%. C₉ H₁₄N₅ O₇ P. 0.25H₂ O requires: C, 31.81, H, 4.27; N, 20.61%. m/z: (positiveion FAB) 336 (M+H⁺).

EXAMPLE 11 2,6-Diamino-9-[2-(diethoxyphosphorylmethoxy)ethoxy]purine

A solution of6-chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]-2-formamidopurine (0.80g, 1.96 mmol) in ethanol (10 ml) saturated with ammonia gas was heatedin a sealed steel vessel at 110° C. for 51/2 hours. The reaction mixturewas cooled to ambient temperature, evaporated to dryness and the residuechromatographed on silica gel (eluted with dichloromethane:methanol90:10) to give the title compound (0.25 g, 35%), m.p. 139°-141°(acetonitrile). UV: ν_(max) (EtOH) 256 (ε8,200), 280 (ε10,200)nm:

IR: ν_(max) (KBr) 3443, 3404, 3320, 3152, 2984, 2940, 1662, 1644, 1608,1586, 1505, 1480, 1414, 1403, 1360, 1338, 1277, 1265, 1241, 1229, 1221,1187, 1163, 1113, 1098, 1052, 1024, 978, 881, 842, 789, 769, 750 cm⁻¹. ¹H NMR: δ_(H) [(CD₃)₂ SO] 1.24 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.81 (2H, m,CH₂), 3.92 (2H, d, J=7.98 Hz, OCH₂ P), 4.06 (4H, m, (OCH₂ CH₃)₂), 4.38(2H, m, N-OCH₂), 5.93 (2H, br.s, D₂ O exchangeable NH₂), 6.79 (2H, br.s,D₂ O exchangeable NH₂), 7.88 (1H, s, H-8).

Found: C,39.57; H,5.82; N,23.27%; C₁₂ H₂₁ N₆ O₅ P. 0.1 H₂ O requiresC,39.80; H,5.90; N,23.21%. m/z: observed 360.1318; C₁₂ H₂₁ N₆ O₅ P(M⁺)requires 360.1312.

EXAMPLE 12 2,6-Diamino-9-[2-phosphonomethoxy)ethoxy]purine

To a solution of2,6-diamino-9-[2-(diethoxyphosphorylmethoxy)ethoxy]purine (0.15 g, 0.41mmol) in dry dichloromethane (3 ml) and dry dimethylformamide (1 ml) wasadded trimethylsilyl bromide (0.55 ml, 4.1 mmol) and the solutionstirred at ambient temperature for 2 hours. The solvent was removedunder reduced pressure and the residue dissolved in methanol. Thesolution was evaporated to dryness and the solid residue obtainedtriturated with methanol to give the title compound (75 mg, 59%)m.p. >300°. IR: ν_(max) (KBr) 3369, 3145, 3138, 3120, 1658, 1617, 1593,1532, 1485, 1455, 1419, 1405, 1368, 1285, 1232, 1163, 1118, 1059, 915,886, 841, 781, 766, 741, 716 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.60 (2H,d, J=8.25 Hz, OCH₂ P), 3.75 (2H, m, CH₂), 4.37 (2H, m, NOCH₂), 6.0 (2H,br.s, D₂ O exchangeable NH₂), 6.82 (2H, br.s, D₂ O exchangeable NH₂),7.93 (1H, s, H-8), 2.5- 5.0 (broad, D₂ O exchangeable PO(OH)₂ +waterfrom solvent). Found: C,30.28; H,4.81; N,26.03%. C₈ H₁₃ N₆ O₅ P. 0.9H₂ Orequires; C,29.99; H,4.65; N,26.23%.

EXAMPLE 13 2-Amino-6-chloro-9-[2-(phosphonomethoxy)ethoxy]purine

To a solution of6-chloro-9-[2-(diethoxyphosphorylmethoxy)ethoxy]-2-formamidopurine(0.370 g, 0.91 mmol) in dry dimethylformamide (5 ml) was addedtrimethylsilyl bromide (1.3 ml, 9.6 mmol) and the solution stirred atambient temperature for 3 hours. The solvent was removed under reducedpressure and the residue co-evaporated with acetone:water 1:1 (×2). Thesolid residue obtained was crystallised from hot water to give the titlecompound as a tan solid (0.15 g, 51%), m.p. 190°-195° C. IR: ν_(max)(KBr) 3480, 3430, 3200, 3150, 2920, 1660, 1625, 1570, 1525, 1480, 1370,1320, 1235, 1195, 1125, 1105, 1030, 995, 950, 920, 880, 780 cm⁻¹. ¹ HNMR: δ_(H) [(CD₃)₂ SO] 3.63 (2H, d, J=8.52 Hz, OCH₂ P), 3.81 (2H, m, CH₂OCH₂ P), 4.45 (2H, m, N-OCH₂), 2.75-5.25 (2H, broad, D₂ O exchangeablePO(OH)₂), 7.11 (2H, br.s, D₂ O exchangeable NH₂), 8.37 (1H, s, H-8).Found: C,28.89; H,3.51; N,21.15%. C₈ H₁₁ N₅ O₅ PCl. 0.5H₂ O requires:C,28.88; H,3.63; N,21.06%.

m/z: (FAB, thioglycerol matrix) 324 (MH⁺, 100%).

EXAMPLE 14 2-Amino-6-methoxy-9-[2-(phosphonomethoxy)ethoxy]purine

To a solution of 6-chloro-9-[2-(phosphonomethoxy)ethoxy]quanine (0.10 g,0.31 mmol) in methanol (3 ml), was added sodium methoxide (0.59 ml of 30wt % solution in methanol, 3.1 mmol) and the reaction mixture stirred at50° C. for 5 hours. The solution was cooled to ambient temperature,diluted with methanol (20 ml) and Amberlite IR-120(plus) resin added. (Asmall excess of the resin was added to obtain a solution of pH 2.5). Thesolution was filtered and the resin washed with methanol:water (1:1, 30ml) and the combined filtrates concentrated under reduced pressure. Theresidue was purified by reverse-phase silica column chromatography usingwater as eluant to give the title compound as a white solid (70 mg,71%). UV: ν_(max) (H₂ O) 248 (ε 7,230), 280 (ε 8250)nm. ¹ H NMR: δ_(H)[(CD₃)₂ SO] 3.38 (2H, d, J=8.35 Hz, OCH₂ P), 3.70 (2H, m, CH₂), 3.95(3H, s, OCH₃), 4.37 (2H, m, NOCH₂), 4.4-5.0 (2H, br.s, D₂ O exchangeablePO(OH)₂), 6.73 (2H, br.s, D₂ O exchangeable NH₂), 8.19 (1H, s H-8).

EXAMPLE 159-[(2-Hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methoxy]quanine

A solution of dicyclohexylcarbodiimide (0.323 g, 1.57 mmol) in t-butanol(15 ml) and DMF (2 ml) was added dropwise over 30 minutes to a solutionof 9-[3-hydroxy-2-(phosphonomethoxy)propoxy]quanine (0.105 g, 0.31 mmol)and N,N-dicyclohexyl-4-morpholinocarboxamidine (0.092 g, 0.31 mmol) in50% aqueous t-butanol (10 ml). The reaction mixture was heated underreflux for 8 hours, allowed to cool to ambient temperature overnight andthen filtered. The filtrate was evaporated to dryness, water added tothe residue and the mixture filtered through a glass fibre filter paper.The filtrate was evaporated to dryness and the solid obtained purifiedon DEAE-Sephadex (HCO₃ ⁻ form) eluting with a linear gradient of aqueoustriethylammonium bicarbonate (pH 7.5) from 0.050M to 0.250M. Therequired component was found in the 0.15M fractions. Relevant fractionswere combined and evaporated to dryness co-evaporating withethanol/water 3:1 (×3) when all traces of triethylamine were removed.The residue was converted into a sodium salt form by passing an aqueoussolution through a column of DOWEX 50W-X8 (Na⁺) resin and eluting withwater. Relevant fractions were combined and lyophilized to give thetitle compound as a white amorphous solid (58 mg) UV: ν_(max) (H₂ O) 253(ε 11,600)nm. IR: ν_(max) (KBr) 3408, 3140, 2956, 1696, 1611, 1529,1479, 1382, 1323, 1239, 1210, 1176, 1082, 1044, 1012, 961, 820, 792, 777cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.40 (1H, dd, J=13.1 Hz, J=3.4 Hz,OCH_(B) P), 3.54 (1H, dd, J=13.1 Hz, J=8.4 Hz, OCH_(A) P), 3.77 (1H, m,CH), 3.91 (1H, ddd, J=11.5 Hz, J=15.0 Hz, J=2.3 Hz, CHCH_(B) OP), 4.11(1H, ddd, J=11.5 Hz, J=9.7 Hz, J=4.3 Hz, CHCH_(A) OP), 4.20 (1H, dd,J=10.6 Hz, J=3.4 Hz, NOCH_(B)), 4.29 (1H, dd, J=10.6 Hz, J=6.6 Hz,NOCH_(A)), 6.85 (2H, br.s, D₂ O exchangeable NH₂), 7.90 (1H, s, H-8),10.99 (1H, br.s, D₂ O exchangeable NH).

EXAMPLE 16 2,6-Diamino-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]purine

A solution of9-[3-acetoxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-chloro-2-formamidopurine(0.16 g, 0.33 mmol) in ethanolic ammonia (5 ml) was heated in a sealedvessel at 120° C. for 5 hours. The solution was allowed to cool toambient temperature and the solvent was removed under reduced pressureto leave a brown residue. The residue was purified by preparative t.l.c.(dichloromethane:methanol 80:20) to give2,6-diamino-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]purine asa colourless gum (0.065 g). The gum (0.06 g, 0.15 mmol) was dissolved indry DMF (1 ml) and trimethylsilyl bromide (0.3 ml, 1.53 mmol) added.After 3 hours at ambient temperature, the solvent was removed underreduced pressure and the residue obtained dissolved in 50% aqueousacetone. Evaporation of the solvent gave a solid which aftercrystallisation from hot water, gave the title compound as a cream solid(0.014 g, 27%). ¹ H NMR: δ_(H) [(CD₃).sub. 2 SO] 3.46-3.60 (2H, m, CH₂OH), 3.65-3.85 (3H, m, CHOCH₂ P), 4.24 (1H, m, NOCH_(B)), 4.42 (1H, m,NOCH_(A)), 6.03 (2H, br. s, D₂ O exchangeable NH₂), 6.88 (2H, br. s, D₂O exchangeable NH₂), 8.02 (1H, s, H-8), 3.3 (br.s, D₂ O exchangeable OH,PO(OH)₂, water in solvent). m/z (FAB, thioglycerol matrix) 335 (MH⁺, 6%)

EXAMPLE 17 (R)-9-3-Hydroxy-2-(phosphonomethoxy)propoxy]quanine

a) To a stirred mixture of diethyl(S)-[1-(t-butyldiphenylsilyloxymethyl)-2-hydroxyethoxy]methylphosphonate(540 mg, 1.12 mmol), triphenylphosphine (440 mg, 1.68 mmol) and2-[bis-(t-butoxycarbonyl)amino]-9-hydroxy-6-methoxypurine (prepared asdescribed in EF-A-319228) (428 mg, 1.12 mmol) in dry THF (20 ml), cooledin ice and under a nitrogen atmosphere, was added dropwise diethylazodicarboxylate (0.27 ml, 1.68 mmol). Within five minutes completedissolution had occurred and the ice bath was removed. After 1 hour, thesolution was evaporated to dryness and the residue obtainedchromatographed on silica gel (initial eluant hexane: ethyl acetate 2:1,then hexane:ethyl acetate in the proportions 1:1, 1:2, 1:3) to yield(S)-2-[bis-(t-butoxycarbonyl)amino]-9-[3-(t-butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]-6-methoxypurineas a colourless glass (310 mg, 32%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 0.96(9H, s, t-Bu), 1.18, 1.19 (2×3H, 2×t, J= 7 Hz, (OCH₂ CH₃)₂), 1.37 (18H,br.s, 2×t-Bu), 3.7-4.15 (12H, m, CHCH₂, (OCH₂ CH₃)₂, OCH₂ P, OCH₃),4.45-4.70 (2H, m, NOCH₂), 7.25-7.75 (10H, m, 2×Ph), 8.75 (1H, s, H-8).m/z: (FAB+ve ion, thioglycerol/HCl) 844 (MH⁺, 4%), 644 (100%).

b) A solution of(S)-2-[bis-(t-butyloxycarbonyl)amino]-9-[3-(t-butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]-6-methoxypurine(300 mg, 0.36 mmol) in 67% aqueous trifluoroacetic acid (3 ml) was keptat ambient temperature for 3 hours. The solution was washed with hexane(3×10 ml) and the aqueous phase evaporated to dryness. The residueobtained was chromatographed on silica gel (dichloromethane: methanol95:5 as eluant, then 90:10) to give(R)-2-amino-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]-6-methoxypurineas a pale yellow glass (101 mg, 70%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23(6H, t, J=7.1 Hz, (OCH₂ CH₃)₂), 3.5-3.65 (2H, m, CH₂), 3.70-3.80 (1H, m,CH), 3.96 (3H, s, OCH₃), 3.98-4.10 (6H, m, (OCH₂ CH₃)₂, OCH₂ P), 4.31(1H, dd, J=11.27 Hz, J=6.87 Hz, NOCH_(B)), 4.45 (1H, dd, J=11.27 Hz,J=3.02 Hz, NOCH_(A)), 4.85 (1H, t, J=5.50 Hz, D₂ O exchangeable OH),6.60 (2H, s, D₂ O exchangeable NH₂), 8.14 (1H, s, H-8). m/z: (FAB+veion, thioglycerol) 406 (MH⁺, 100%).

c) To a solution of(R)-2-amino-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy)-6-methoxypurine(100 mg, 0.25 mmol) in dry dimethylformamide (1 ml) was addedtrimethylsilyl bromide (0.5 ml, 3.8 mmol) and the solution stirred atambient temperature for two hours. The solvent was removed under reducedpressure and the residue co-evaporated with acetone-water, 1:1 (×2). Thesolid obtained was crystallised from hot water to give the titlecompound as a cream solid (16 mg, 20%). ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.52(2H, m, CH₂ OH), 3.60-3.80 (3H, m, OCH₂ P, CH), 4.25 (1H, dd, J=11.9 Hz,J=7.1 Hz, NOCH_(B)), 4.40 (1H, dd, J=11.9 Hz, J=3.3 Hz, NOCH_(A)), 6.60(2H, br.s, D₂ O exchangeable NH₂), 8.00 (1H, s, H-8), 10.65 (1H, br.s,D₂ O exchangeable NH), 2.6-4.1 (3H, broad, D₂ O exchangeable OH,PO(OH)₂). m/z: (FAB+ve ion, thioglycerol) 336 (MH⁺, 28%).

EXAMPLE 18(S)-9-[(2-Hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methoxy]quanine

A solution of dicyclohexylcarbodiimide (0.615 g, 3.0 mmol) in t-butanol(15 ml) was added dropwise over 30 minutes to a solution of(R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]quanine (0.20 g, 0.6 mmol)and N,N-dicyclohexyl-4-morpholinocarboxamidine (0.175 g, 0.6 mmol) in50% aqueous t-butanol (30 ml). The reaction mixture was heated underreflux for 51/2 hours, allowed to cool to ambient temperature andevaporated to dryness. Water was added to the residue and the resultingmixture filtered through a glass fibre filter paper. The filtrate wasevaporated to dryness and the solid obtained purified on DEAE-Sephadex(HCO₃ ⁻ form) eluting with a linear gradient of aqueous triethylammoniumbicarbonate (pH 7.5) from 0.001M to 0.25M. Relevant fractions werecombined and evaporated to dryness co-evaporating with ethanol/water 3:1(×3) to remove all traces of triethylamine. The product was convertedinto the sodium salt using DOWEX 50W-X8 (Na⁺ -form). The title compoundwas obtained as a white powder (0.16 g, 79%) after lyphilisation.

UV: ν_(max) (H₂ O) 253 (ε 11,180)nm. IR: ν_(max) (KBr) 3411, 3400, 3144,2759, 1696, 1617, 1591, 1476, 1380, 1330, 1239, 1209, 1174, 1081, 1045,1010, 96, 815, 796 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.40 (1H, dd, J=12,9Hz, J=3.3 Hz, OCH_(B) P) 3.50 (1H, dd, J=12.9 Hz, J=8 Hz, OCH_(A) P),3.74 (1H, m, CH), 3.90 (1H, m, CH_(B) OP), 4.0-4.3 (3H, m, CH_(A) OP,NOCH₂), 6.75 (2H, br.s, D₂ O exchangeable NH₂), 7.90 (1H, s, H-8), 10.81(1H, br.s, D₂ O exchangeable NH). m/z: (FAB, thioglycerol matrix) 340(MH⁺). [α]_(D) ²⁵ (H₂ O) - 38.7° (c=0.93).

EXAMPLE 19 (R)-9-[3-Hydroxy-2-(phosphonomethoxy)propoxy]adenine

a) To a stirred mixture of diethyl(S)-[1-(t-butyldiphenylsilyloxymethyl)-2-hydroxyethoxy]methylphosphonate(1.07 g, 2.2 mmol), triphenylphosphine (0.7 g, 2.7 mmol) and9-hydroxy-6-phthalimidopurine (prepared as described in EP-A-319228)(0.63 g, 2.2 mmol) in dry THF (20 ml), cooled in ice and under anitrogen atmosphere, was added dropwise diethyl azodicarboxylate (0.42ml, 2.7 mmol). The resulting solution was stirred overnight at ambienttemperature, evaporated to dryness and the residue obtainedchromatographed on silica gel using hexane/ethyl acetate 3:1 as eluant.The eluant was changed as follows: hexane/ethyl acetate 1:1,hexane/ethyl acetate 1:2 and finally to ethyl acetate to give(S)-9-[3-(t-butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurineas a cream foam (1.1 g, 66%). IR: _(max) (film) 3070, 2930, 2860, 1795,1603, 1580, 1470, 1450, 1430, 1405, 1365, 1330, 1250, 1175, 1160, 1105,1020, 970, 885, 835, 795, 780 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 0.97 (9H,s, t-Bu), 1.19, 1.20 (2×3H, 2×t, J=7 Hz, (OCH₂ CH₃)₂), 3.8-4.15 (9H, m,(OCH₂ CH₃)₂, OCH₂ P, CHCH₂), 4.65 (1H, dd, J=11.5 Hz, J=6.6 Hz,NOCH_(B)), 4.78 (1H, dd, J=11.5 Hz, J=3.0 Hz, NOCH_(A)), 7.3-7.7 (10H,m, 2×Ph), 8.0-8.15 (4H, m, aromatic H), 9.04 (1H, s, H-2 or H-8), 9.08(1H, s, H-2 or H-8). Found: C, 59.51; H, 5.76; N, 9.42%. C₃₇ H₄₂ N₅ O₈PSi requires: C, 59.74; H, 5.69; N, 9.41%. m/z: (FAB, thioglycerolmatrix) 744 (MH⁺).

b) A solution of(S)-9-[3-(t-butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurine(0.98 g, 1.6 mmol) in dry dichloromethane (10 ml) was treated withN-methylhydrazine (0.093 ml, 1.75 mmol) at room temperature for 1 hour.The reaction mixture was filtered, the filtrate concentrated in vacuoand the residue chromatographed on silica gel using dichloromethane asthe initial eluant. The eluant was changed to dichloromethane/methanol96:4 to give(S)-9-[3-(t-butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]adenineas a colourless oil (0.61 g, 75%). UV: ν_(max) (EtOH) 260 (13,690) nm.IR: ν_(max) (film) 3322, 3176, 2930, 2902, 2857, 1643, 1594, 1471, 1427,1405, 1398, 1325, 1292, 1251, 1112, 1051, 1026, 971, 823, 795, 741 cm⁻¹.¹ H NMR: δ_(H) [(CD₃)₂ SO] 0.94 (9H, s, t-Bu), 1.19 (2×3H, 2×t, J=7 Hz,(OCH₂ CH₃)₂), 3.7-4.15 (9H, m, CHCH₂, OCH₂ P, (OCH₂ CH₃)₂), 4.48 (1H,dd, J=11.27 Hz, J=6 Hz, NOCH_(B)), 4.65 (1H, dd, J=11.27 Hz, J=2.75 Hz,NOCH_(A)), 7.3-7.7 (12H, m, 2×Ph+D₂ O exchangeable NH₂), 8.14 (1H, s,H-2 or H-8), 8.41 (1H, s, H-2 or H-8). Found: C, 55.65; H, 6.52; N,11.24%. C₂₉ H₄₀ N₅ O₆ PSi 0.75 H₂ O requires: C, 55.53; H, 6.67; N,11.16%. m/z: (FAB, thioglycerol matrix) 614 (MH⁺).

c)(S)-9-[3-(t-Butyldiphenylsilyloxy)-2-(diethoxyphosphorylmethoxy)propoxy]adenine(0.61 g, 1 mmol) was dissolved in trifluoroacetic acid/water, 2:1 (6 ml)and the solution stirred at ambient temperature for 3 hours. Hexane (10ml) was added and the mixture shaken. The aqueous phase was separated,washed once more with hexane (10 ml) and evaporated to dryness. Theresidue was treated with ethanolic ammonia solution (5 ml), at ambienttemperature, for 5 minutes after which the solution was evaporated todryness. The residue was chromatographed on silica gel usingdichloromethane/methanol 90:10 as eluant to give(R)-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenine as acolourless oil (0.3 g, 80%) which on prolonged standing, crystallised aswhite needles m.p. 98°-102°. IR: ν_(max) (film) 3340, 3210, 2995, 2930,2910, 1660, 1645, 1600, 1470, 1445, 1415, 1395, 1370, 1330, 1300, 1240,1210, 1165, 1125, 1045. 1020, 980, 825, 95, 735 cm⁻¹. ¹ H NMR: δ_(H)[(CD₃)₂ SO] 1.23 (6H, t, J=7 Hz (OCH₂ CH₃)₂), 3.5-3.7 (2H, m, CH₂ OH),3.75-3.85 (1H, m, CH), 3.95-4.15 (6H, m, (OCH₂ CH₃)₂, OCH₂ P), 4.38 (1H,dd, J=11.3 Hz, J=6.6 Hz, NOCH_(B)), 4.55 (1H, dd, J= 11.3 Hz, J=3.0 Hz,NOCH_(A)), 4.88 (1H, t, J=5.5 Hz, D₂ O exchangeable OH), 7.38 (2H,br.s., D₂ O exchangeable NH₂), 8.15 (1H, s, H-2 or H-8), 8.43 (1H, s,H-2 or H-8). Found: C, 39.26; H, 6.05; N, 17.33%. C₁₃ H₂₂ N₅ O₆ P 1.25H₂ O requires: C, 39.25; H, 6.20; N, 17 61%. m/z: (FAB, thioglycerolmatrix) 376 (MH⁺).

d) To a solution of(R)-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy)adenine (0.16 g,0.43 mmol) in dry DMF (3 ml) was added trimethylsilyl bromide (0.56 ml,4.3 mmol) and the solution stirred at ambient temperature for 4 hours.The solvent was removed under reduced presence and the residueco-evaporated with acetone/water 1:1 (x3). The solid residue wascrystallised from acetone/water (0.1 g, 77%), m.p. 200°-205°. UV:λ_(max) (EtOH), 260 (11,800) nm. IR: ν_(max) (KBr) 3449, 3400, 3199,3105, 2951, 2897, 1711, 1680, 1612, 1569, 1467, 1420, 1355, 1330, 1305,1244, 1216, 1128, 1080, 924, 866 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)2SO]3.54(2H, m, CH₂ OH), 3.70-3.85 (3H, m, OCH₂ P, CH), 4.36 (1H, dd, J=11 Hz,J=7 Hz, NOCH_(B)), 4.55 (1H, dd, J=11 Hz, J=3 Hz, NOCH_(A)), 7.51 (2H,br.s, D₂ O exchangeable NH₂), 8.17 (1H, s, H-2 or H-8), 8.50 (1H, s, H-2or H-8), 3.5-7.0 (3H, broad, PO(OH)₂, OH). Found: C, 32.07; H, 4.73; N,20.46%. C₉ H₁₄ N₅ O₅ P 1 H₂ O requires: C, 32.05; H, 4.78; N, 20.76%.m/z: (FAB, thioglycerol matrix) 320 (MH⁺). [α]_(D) ²⁵ (0.1M NaOH)-11.5°(c=0.2).

EXAMPLE 20 (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propoxy]guanine

a) To a stirred solution of diethyl(R)-[2-benzyloxy-1-(hydroxymethyl)ethoxy]methylphosphonate (1.0 g, 3mmol), triphenylphosphine (1.18 g, 4.5 mmol) and2-[bis-(t-butoxycarbonyl)amino]-9-hydroxy-6-methoxypurine (1.15 g, 3mmol) in dry THF (20 ml), cooled in ice and under a mitrogen atmosphere,was added, dropwise, diethyl azodicarboxylate (0.71 ml, 4.5 mmol). Theresulting solution was allowed to warm to ambient temperature and leftstirring for 16 hours. The solvent was evaporated under reduced pressureand the residue obtained chromatographed on silica gel usinghexane/ethyl acetate 2:1 as eluant. The eluant was changed as follows:hexane/ethyl acetate 1:1, hexane/ethyl acetate 1:2 and finally to ethylacetate to give(S)-2-[bis-(t-butoxycarbonyl)amino]-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-methoxypurineas a colourless oil (1.85 g, 88%).

IR: ν_(max) (film) 3070, 2990, 2940, 2910, 1795, 1755, 1740, 1595, 1480,1455, 1425, 1395, 1370, 1325, 1255, 1160, 1120, 1100, 1050, 1025, 975,855, 795, 740, 705cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.20 (6H, t, J=7 Hz,(OCH₂ CH₃)₂), 1.38 (18H, s, 2xt-Bu), 3.5-3.75 (2H, m, CH₂), 3.95-4.1(7H, m, OCH₂ P, CH, (OCH₂ CH₃)2), 4.08 (3H, s, OCH₃), 4.45-4.65 (2H, m,NOCH₂) 4.51 (2H, s, OCH₂ Ph), 7.25-7.40 (5H, m, Ph), 8.75 (1H, s, H-8).Found: C, 52.27; H, 6.77; N, 9.65%. C₃₁ H₄₅ N₅ O₁₁ P 1H₂ O requires: C,52.17; H, 6.77; N, 9.81%. m/z: (FAB, TDE/Na matrix) 718 (32%, M+Na⁺) 696(13%, MH⁺).

b) Trifluoroacetic acid (2 ml) was added to an ice cooled solution of(S)-2-[bis-(t-butoxycarbonyl)amino]-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy]propoxy]-6-methoxypurine(1.65 g, 2.3 mmol) in dry dichloromethane (30 ml) an the resultingsolution left for 2 hours. After warming to ambient temperature, 10%Pd-C (800 mg) was added and the mixture hydrogenated at ambienttemperature and pressure for 2 hours. The reaction mixture was filtered,the catalyst washed with dichloromethane (20 ml), the filtrate washedwith saturated aqueous sodium bicarbonate solution, brine, dried (MgSO₄)and evaporated to leave a colourless viscous oil. Chromatography of theoil on silica gel using dichloromethane/methanol 95:5 as the initialeluant, then changing the eluant to dichloromethane/methanol 90:10 gave(S)-2-amino-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]-6-methoxypurineas a colourless oil (0.65 g, 67%). IR: ν_(max) (film) 3360, 3230, 2990,2940, 2910, 1620, 1585, 1500, 1485, 1455, 1390, 1335, 1265, 1240, 1165,1120, 1060, 1025, 975, 825, 785 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.23(6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.45-3.60 (2H, m, collapses to d on D₂ Oexchange, CH₂ OH), 3.70-3.85 (1H, m, CH), 3.96 (3H, s, OCH₃), 3.9-4.15(6H, m, (OCH₂ CH₃, OCH₂ P), 4.31 (1H, dd, J=11.3 Hz, J=6.9 Hz,NOCH_(B)), 4.45 (1H, dd, J =11.3 Hz, J=3 Hz, NOCH_(A)), 4.84 (1H, t,J=5.5 Hz, D₂ O exchangeable OH), 6.59 (2H, br.s, D₂ O exchangeable NH₂),8.14 (3H, s, H-8). m/z: (FAB, thioglycerol matrix) 406 (MH⁺).

c) To a solution of(S)-2-amino-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]-6-methoxypurine(0.56 g, 1.38 mmol) in dry dimethylformamide (5 ml) was addedtrimethylsilyl bromide (1.82 ml, 13.8 mmol) and the solution stirred atambient temperature for 3 hours. The solvent was removed under reducedpressure and the residue co-evaporated with acetone/water 1:1 (x2). Thewhite solid obtained was crystallised from hot water to give the titlecompound as a white solid (0.305 g, 66%). IR:ν_(max) (KBr) 3380, 3320,3160, 1715, 1645, 1600, 1385, 1185, 1160, 1100, 1060, 1045, 925, 770cm⁻¹. ¹ H NMR: δ_(H) (D₂ O+NH₃) 3.61 (1H, dd, J=12.1 Hz J=9.9 Hz, CH_(B)P), 3.65-3.75 (2H, m, CH_(A) P, CH_(B) OH), 3.82-3.89 (1H, m, CH), 3.93(1H, dd, J=12.3 Hz, J=3.6 Hz, CH_(A) OH), 4.46-4.53 (2H, m, NOCH₂), 8.03(1H, s, H-8). Found: C, 32.24; H, 4.16; N, 20.59%. C₉ H₁₄ N₅ O₇ Prequires: C, 32.24; H, 4.21; N, 20.89%. m/z: (FAB, thioglycerol matrix)336 (MH⁺). [α]_(D) ²⁵ (0.1M NaOH)+12.8° (c=0.43).

EXAMPLE 21(R)-9-[(2-Hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)methoxy]guanine

A solution of dicyclohexylcarbodiimide (0.615 g, 3.0 mmol) in t-butanol(10 ml) was added dropwise over 30 minutes to a solution of(S)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]guanine (0.20 g, 0.6 mmol)and N,N-dicyclohexyl-4-morpholinocarboxamidine (0.175 g, 0.6 mmol) in50% aqueous t-butanol (30 ml). The reaction mixture was heated underreflux for 4 hours, allowed to cool to ambient temperature andevaporated to dryness. Water was added to the residue and the mixturefiltered through a glass fibre filter paper. The filtrate was evaporatedto dryness and the solid obtained purified on DEAE-Sephadex (HCO₃ ⁻form) eluting with a linear gradient of aqueous triethylammoniumbicarbonate (pH 7.5) from 0.001M to 0.25M. Relevant fractions werecombined and evaporated to dryness co-evaporating with ethanol/water,3:1 (x2) to remove all traces of triethylamine. The product wasconverted into the sodium salt using DOWEX 50W-X8 (Na⁺ form). The titlecompound was obtained as a white solid (0.12 g, 59%) afterlyophilisation. IR: ν_(max) (KBr) 3400, 3140, 1690, 1610, 1475, 1380,1240, 1208, 1079, 1040, 1010, 960, 790 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO]3.42 (1H, dd, J=13.2 Hz, J=3.3 Hz, OCH_(B) P), 3.55 (1H, dd, J=13.2 Hz,J= 8 Hz, OCHAP), 3.79 (1H, m, CH), 3.92 (1H, m, CH_(B) OP), 4.07-4.32(3H, m, CH_(A) OP, NOCH₂), 6.86 (2H, br.s, D₂ O exchangeable NH₂), 7.90(1H, s, H-8), 10.97 (1H, br.s, D₂ O exchangeable NH). m/z: (FAB,thioglycerol) 318 (M-Na+2H⁺), 340 (MH⁺).

EXAMPLE 22 (S)-9-[3-Hydroxy-2-(phosphonomethoxy)propoxy]adenine

a) To a stirred solution of diethyl(R)-[2-benzyloxy-1-(hydroxymethyl)ethoxy]methylphosphonate (0.9 g, 2.7mmol), triphenylphosphine (1.06 g, 4 mmol) and9-hydroxy-6-N-phthalimidopurine (0.76 g, 2.7 mmol) (prepared as inEP-A-319228) in dry THF (15 ml), cooled in ice and under a nitrogenatmosphere, was added, dropwise, diethyl azodicarboxylate (0.6 ml, 4mmol). The resulting solution was allowed to warm to ambient temperatureand stirred for 4 hours. The solvent was evaporated under reducedpressure and the residue obtained chromatographed on silica gel usinghexane/ethyl acetate 1:1 as the initial eluant. The eluant was changedto ethyl acetate to give(S)-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurineas a pale brown oil (1.16 g, 72%) which was used directly in the nextstage.

b) A solution of(S)-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]-6-N-phthalimidopurine(0.83 g, 1.39 mmol) in dry dichloromethane (10 ml) was treated withN-methylhydrazine (0.08 ml, 1.53 mmol) at ambient temperature for 3hours. The reaction mixture was filtered, the filtrate concentrated invacuo and the residue chromatographed on silica gel usingdichloromethane/methanol 98:2 as eluant to give(S)-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine as acolourless gum (0.27 g, 41%). IR: ν_(max) (film) 3320, 3180, 2990, 2905,1650, 1595, 1470, 1455, 1410, 1390, 1370, 1295, 1245, 1160, 1095, 1050,1025, 970, 820, 790, 730, 700 cm⁻¹. ¹ H NMR: δ_(H) [(CD₃)₂ SO] 1.21(2x3H, 2xt, J=7 Hz, (OCH₂ CH₃)₂), 3.61-3.72 (2H, m, CH₂ OCH₂ Ph),3.96-4.12 (7H, m, CH, OCH₂ P, (OCH₂ CH.sub. 3)₂), 4.40-4.58 (2H, m,NOCH₂), 4.52 (2H, s, OCH₂ Ph), 7.25-7.50 (7H, m, Ph+D₂ O exchangeableNH₂), 8.15 (1H, s, H-2 or H-8), 8.41 (1H, s, H-2 or H-8).

c) 10% Pd-C (50 mg) was added to a solution of(S)-9-[3-benzyloxy-2-(diethoxyphosphorylmethoxy)propoxy]adenine (0.21 g,0.45 mmol) in dichloromethane (5 ml) and trifluoroacetic acid (lml) andthe mixture hydrogenated at atmospheric pressure and ambient temperaturefor 5 hours. The mixture was filtered, the filtrate evaporated and theresidue obtained dissolved in ethanolic ammonia solution (5 ml). After15 minutes the solvent was evaporated and the residue chromatographed onsilica gel using dichloromethane/methanol 90:10 as eluant. T.l.c.examination of the product indicated traces of a non UV absorbingimpurity which was removed by prep. t.l.c. usingdichloromethane/methanol 88:12 as eluant.(S)-9-[2-(diethoxyphosphorylmethoxy)3-hydroxypropoxy]adenine wasobtained as a colourless gum (0.11 g, 65%). ¹ H NMR: δ_(H) [(CD₃)₂ SO]1.23 (6H, t, J=7 Hz, (OCH₂ CH₃)₂), 3.5-3.65 (2H, m, CH₂ OH), 3.75-3.85(1H, m, CH), 3.98-4.15 (6H, m, OCH₂ P, (OCH₂ CH₃)₂), 4.38 (1H, dd, J=6.6Hz, J=11.3 Hz, NOCH_(B)), 4.54 (1H, dd, J=3 Hz, J=11.3 Hz, NOCH_(A)),4.88 (1H, t, J=5.5 Hz, D₂ O exchangeable OH), 7.38 (2H, br.s, D₂ Oexchangeable NH₂), 8.15 (1H, s, H-2 or H-8), 8.43 (1H, s, H-2 or H-8).m/z: (FAB+, thioglycerol matrix) 376 (MH⁺).

d) To a solution of(S)-9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenine (0.09 g,0.24 mmol) in dry dichloromethane (3 ml) was added trimethylsilylbromide (0.31 ml, 2.4 mmol) and the solution stirred at ambienttemperature for 4 hours. The solvent was removed under reduced pressureand the residue co-evaporated with acetone/water 1:1 (x3).Crystallisation of the residue from water/acetone gave the titlecompound as a white solid (0.06 g, 78%), mp. 93°-198° C.

¹ H NMR: δ_(H) [(CD₃)₂ SO] 3.55 (2H, m, CH₂ OH), 3.65-3.85 (3H, m, OCH₂P, CH), 4.36 (1H, dd, J=7 Hz, J=11 Hz, NOCH_(B)), 4.55 (1H, dd, J=3 Hz,J=11 Hz, NOCH_(A)), 7.61 (2H, br.s, D₂ O exchangeable NH₂), 8.19 (1H, s,H-2 or H-8), 8.52 (1H, s, H-2 or H-8), 2.7-5.5 (3H, broad, D₂ Oexchangeable PO(OH)₂, OH).

Antiviral Activity

1. Plaque Reduction Test for Herpes Simplex Viruses 1 and 2

Vero or MRC-5 cells were grown to confluence in 24 well multi-dishes(well diameter=1.5 cm). The drained cell monolayers were each infectedwith approximately 50 infectious particles of herpes simplex virus 1(HSV-1; HFEM strain) or herpes simplex virus 2 (HSV-2; strain MS) in 100μl of phosphate-buffered saline. The virus was adsorbed for 1 hour atroom temperature. After adsorption, residual inoculum was removed fromeach well and replaced with 0.5 ml of Eagle's MEM containing 5% newborncalf serum and 0.9% agarose (A37). Once the agarose had set, dilutionsof the test compound, which had been prepared in Eagle's MEM (containing5% newborn calf serum), were added, each well receiving 0.5 ml of liquidoverlay. The test compound was diluted to give the following series ofconcentrations: 200, 60, 20, 6 . . . 0.06 μg/ml; final concentrations inthe assay ranged, therefore, between 100μg/ml and 0.03μg/ml. Theinfected cultures were incubated at 37° C. in a uumidified atmosphere of5% CO₂ until plaques were clearly visible (2 or 3 days for Vero cells,usually 1 day for MRC-5 cells).

2. Plaque Reduction Test for Varicella Zoster Virus

MRC-5 cells were grown to confluence in 24 well multi-dishes (welldiameter=1.5cm). The drained cell monolayers were each infected withapproximately 50 infectious particles of varicella zoster virus (VZV;Ellen strain) in 100 μl of phosphate-buffered saline. The virus wasadsorbed for 1 hour at room temperature. After adsorption, residualinoculum was removed from each well and replaced with 0.5 ml of Eagle'sMEM containing 5% heat-inactivated foetal calf serum and 0.9% agarose(A37). Once the agarose had set, dilutions of the test compound, whichhad been prepared in Eagle's MEM (containing 5% heat-inactivated foetalcalf serum), were added, each well receiving 0.5 ml of liquid overlay.The test compound was diluted to give the following series ofconcentrations: 200, 60, 20, 6. . . 0.06 μg/ml; final concentrations inthe assay ranged, therefore, between 100 μg/ml and 0.03 μg/ml. Theinfected cultures were incubated at 37° C. in a humidified atmosphere of5% CO₂ until plaques were clearly visible (5 or 6 days).

Cultures from 1 and 2 were fixed in formal saline, the agarose overlayswere carefully washed off, and then the cell monolayers were stainedwith carbol fuchsin. A stereo microscope wasused to count plaques. TheIC₅₀ (concentration of drug which inhibits the number of plaques formedby 50% relative to the number of plaques observed in virus controlmonolayers) of the test compound was calculated. In addition, themonolayers were examined for evidence of drug-induced cytotoxicity; theminimum concentration at which cytotoxicity occurred was recorded.

3. CPE Inhibition Test (Established Monolayer) for Lentiviruses

3×10⁴ sheep choroid plexus (SCP) cells were plated into individual wellsof a 96 well microtitre plate in 100 μl of Eagle's MEM with Hanks' saltscontaining 10% heat inactivated foetal calf serum (FCS). When monolayershad become established (after 1 or 2 days growth) they were washed with200 μl of maintenance medium (Eagle's MEM with Hanks'salts containing0.5% FCS) and infected with 100 μl of visna virus (strain K184) inmaintenance medium (20 TCID₅₀ /ml). Test samples were diluted withmaintenance medium in further 96 well microtitre plates over the range200-0.06 μg/ml by 3-fold dilution steps. 100 μl of the diluted sampleswas then transferred directly onto virus-infected monolayers (finalconcentration range therefore 100-0.03 μg/ml) and incubated at 37° C. ina humidified atmosphere containing, 5% CO₂ until virus-induced CPE wasmaximal in the untreated virus-infected controls (usually 12-14 days).The plates were fixed with formal saline and stained with crystalviolet. Virus-induced CPE was then scored microscopically and theminimum concentration of sample giving complete protection of the cellmonolayers (MIC) determined.

4. Plaque Reduction Test for Cytomegalovirus

MRC-5 cells were grown to confluence in 24 well multi-dishes (welldiameter=1.5 cm). The drained cell monolayers were each infected withapproximately 50 infectious particles of cytomegalovirus (CMV; AD-169strain) in 100 μl of phosphate-buffered saline. The virus wasadsorbedfor 1 hour at room temperature. After adsorption, residual inoculum wasremoved from each well and replaced with 1 ml of Eagle's MEM containing10% heatinactivated foetal calf serum and 0.9% agarose (A37). Once theagarose had set, dilutions of the test compound, which had been preparedin Eagle's MEM (containing 10% heat-inactivated calf serum), were added,each well receiving 1 ml of liquid overlay. The test compound wasdiluted to give the following series of concentrations: 200, 60, 20, 6 .. . 0.06 μg/ml; final concentrations in the assay range, therefore,between 100 μg/ml and 0.03 μg/ml. The infected cultures were incubatedat 37° C. in a humidified atmosphere containing 55 CO₂ until plaqueswere clearly visible (about 12 days). The cultures are fixed in formolsaline, the agarose overlays were carefully washed off, and then thecell monolayers were stained with carbol fuchsin. A stereo microscopewas used to count plaques. The IC₅₀ (concentration of drug whichinhibits the number of plaques formed by 50% relative to the number ofplaques observed in virus control monolayers) of the test compound wascalculated. In addition, the monolayers were examined for evidence ofdrug-induced cytotoxicity; the minimum concentration at whichcytotoxicity occurs was recorded.

5. Test for Human Immunodeficiency Virus (HIV)

a) Cell cytotoxicity test

Peripheral human lymphocytes were isolated by density gradientcentrifugation from blood donations of healthy volunteers. The `buffycoat` fractions of these donations were provided by blood donationcentres.

The buffy coat was diluted 1:1 with sterile phosphate buffered saline(PBS; 50 mM sodium phosphate, pH 7.4, 0,9% sodium chloride) andsubsequently layered over Ficoll. Following centrifugation (30 minutesat 400 x g) the supernatant was discarded and the interphase containingthe lymphocytes was recovered. The lymphocytes were washed two times inPBS and were resuspended finally in cell culture medium.

A viability staining was performed by means of the trypan bluedye-exclusion method. The concentration of cells in the suspension andthe percentage of viable cells were calculated. Subsequently, the cellsuspension was adjusted to a concentration of 1×10⁷ cells/ml. This cellsuspension was transferred to tissue culture flasks: Two thirds of thecell suspension were polyclonally activated by addition ofphytohemagglutinin (final concentration 5 μg.ml). One third of the cellsuspension remained unstimulated.

The lymphocytes were cultivated in an incubator with a humidifiedatmosphere and 5% CO₂ for 48 to 64 hours at 37° C. Following thisincubation period, cells were harvested by centrifugation, resuspendedin cell culture medium and counted. Stimulated and unstimulated cellswere combined in a ratio of 2:1 and adjusted to a concentration of 2×10⁶cells/ml with cell culture medium that contained, in addition, 10units/ml of human recombinant interleukin-2.

Only those preparations of lymphocytes were employed for the screeningtest in which more than 70% of the stimulated lymphocytes expressed theCD 25 antigen and more than 45% of the lymphocytes expressed the CD 4antigen.

100 ug of this lymphocyte suspension was added to each well ofmicrotiter plates containing the test compounds serially diluted overthe range 100 μM to 0.1 μM. Subsequently, the microtiter plates werecultivated for 4 days at 37° C.

Survival and proliferation of the lymphocytes grown in the presence ofthe compound were measured by a quantitative colorimetric assay. Viablecells cultivated in the presence of the dye MTT[3-4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium) reduce this paleyellow substrate by activity of their mitochondrial dehydrogenases to apurple formazan. The amount of product which is a function of cellnumber and metabolic cellular activity was quantified photometrically.By this assay, potential cytotoxic and cytostatic effects of compoundstowards lymphocytes were detected precisely.

Microtiter plates were centrifuged for 5 minutes at 900×g. Thesupernatant was discarded and the cells of each well were resuspended in50 μl of cell culture medium containing 2 mg/ml of MTT. After four hoursof incubation at 37° C. 100 μl of solvent (isopropanol with 0.04 N HCland 10% (v/v) Triton 100) was added to each well. By shaking themicrotiter plates the formazan was solubilized. Subsequently, the plateswere evaluated in an ELISA photometer in the dual wavelength mode(measuring wavelength: 550 nm; reference wavelength: 690 nm).

For each well the difference in absorption (abs. 550 nm - abs. 690 nm)was calculated. These data provided the basis for further evaluation ofthe cytotoxicity test. The approximate CD₅₀ (halfmaximal cytotoxic dose)of each compound was calculated.

b) HIV Suppression test

Peripheral human lymphocytes were prepared, cultivated, and harvested asabove. Following the determination of the lymphocyte surface markers,unstimulated and mitogen stimulated cells were combined in a ratio of1:2.

Under safety conditions these cells are infected with a standardpreparation of HIV. The cells are sedimented by centrifugation. Thesupernatant was discarded and cells were resuspended in the HIVinoculum.

This inoculum is a liquid suspension of HIV-1 strain virus, pretestedand adjusted to a titer that results in a synthesis of viral coreprotein p24 of >100 ng/ml at day four following infection of humanlymphocytes according to the protocol.

3×10⁸ lymphocytes were resuspended in 1 ml HIV inoculum and incubated at37° C. for 60 minutes. Subsequently, the cells were washed two timeswith 50 ml of culture medium and resuspended in culture mediumcontaining 10 units/ml of human recombinant interleukin-2 to yield acell concentration of 2×10⁶ cells/ml. 100 μl of this cell suspension wasadded to each well of the microtiter plates containing the dilutedsolutions of the compounds. The microtiter plates were cultivated in anincubator with a humidified atmosphere and 5% CO₂ at 37° C.

Accordingly, a fraction of lymphocytes was mock-infected with the samevirus preparation that was heat inactivated (30 minutes at 56° C.) priorto infection.

On each of the days 2,3 and 4 post infection one of the microtiterplates which had been established in triplicate was prepared fordetermination of viral replication. Viral RNA is determined within thecells whereas the viral core protein p24 was detected in the supernatantof the lymphocyte culture.

Accordingly, 150 μl of supernatant were removed from each well andtransferred to the well of a microtiter plate containing 50 μl well ofSDS (sodium dodecylsulfate, 0.08%). These plates were stored frozen. 50μl of stop solution (1% SDS, 20 mM sodium acetate, pH 5.0, and 200 μg/mlheparin) were added to the cells remaining in each well. The plates werestored frozen.

The concentration of p24 synthesized by the HIV infected cells wasdetermined by means of a sandwich ELISA, while the concentration ofviral RNA was quantitated by nucleic acid hybridisation, using a ^(32p)-labelled DNA probe for the gag/pol region of the viral genome. Absolutelevels of viral antigen and RNA in drug treated samples were comparedwith untreated, virus-infected controls and the percentage inhibitioncalculated.

    ______________________________________                                        Results                                                                       IC.sub.50 (μg/ml)                                                          Herpes Simplex                MIC (μg/ml)                                  virus           Varicella     Visna                                           Type 1      Type 2  Zoster virus  Virus                                       HFEM        MS      Ellen         K184                                        strain      strain  strain        strain                                      in          in      in            in                                          Example                                                                              Vero     MRC-5   MRC-5       SCP                                       No.    cells    cells   cells       cells                                     ______________________________________                                        1      >100     >100    >100        100                                       2      >100     69      80          3                                         3      >100     >100    51          10                                        4      1.1      0.08    0.06        <0.003                                    5      59       --      <3          --                                        6      >100     >100    66          0.3                                       8      >100     >100    60          1                                         10     17       7.3     21          <0.003                                    11     >100     --      >100        30                                        12     24       --      <3          0.3                                       13     3.7      --      0.5         <0.03                                     14     <3       --      <0.03       <0.03                                     15     29       --       <3         --                                        17     7        10      17                                                    18     21       15      13                                                    20     25       23      52                                                    21     73       36      24                                                    ______________________________________                                    

At concentrations up to 30 μg/ml, none of the compounds were cytotoxicfor the cell monolayers used in any of the above the tests.

Against CMV virus, the compounds of Examples 17, 18, 20 and 21 gave IC₅₀values of 3, 3, 29 and 17 μg/ml respectively.

Against HIV virus, the following results were obtained.

    ______________________________________                                                     % Inhibition on Days 3 and 4                                                  after infection                                                               Viral Antigen                                                                            Viral RNA                                             Ex. No. Concn. (μM)                                                                           Day 3    Day 4 Day 3  Day 4                                ______________________________________                                         2      10          89       91   87     93                                    4      10         100       99   99     95                                    7      10          95       77   95     64                                   10       1          11       44   17     53                                   12      10          77      100   47     100                                  13      10          99      100   96     97                                   14      10         100      100   100    99                                   15       1         100       52   72     65                                   17       1         100       35   73     54                                   18      10         100       33   92     32                                   19      10          80       91   83     90                                   20       1          54       15   10     14                                   21      10          50       7    64     0                                    ______________________________________                                    

We claim:
 1. A compound of formula (I), or a pharmaceutically acceptablesalt thereof: ##STR13## wherein R₁ is hydroxy, amino, chloro or OR₇whereinR₇ is C₁₋₆ alkyl, phenyl or phenyl C₁₋₂ alkyl either of whichphenyl moieties may be substituted by one or two substsituents selectedfrom halo, C₁₋₄ alkyl or C₁₋₄ alkoxy; R₂ is amino or, when R₁ is hydroxyor amino, R₂ may also be hydrogen; R₃ is hydrogen, hydroxymethyl oracyloxymethyl wherein the acyl moiety os C₁₋₇ alkanoyl or optionallysubstituted benzoyl; R₄ is a group of formula: ##STR14## wherein R₅ andR₆ are independently selected from hydrogen, C₁₋₆ alkyl and optionallysubstituted phenyl; or R₃ and R₄ together are: ##STR15## wherein R₆ isas defined above; andwherein the optional substituents for the phenyl orbenzoyl are 1-3 groups or atoms selected from halogen, C₁₋₄ alkyl, andC₁₋₄ alkoxy.
 2. A compound according to claim 1 wherein R₁ is hydroxyand R₂ is amino.
 3. A compound according to claim 1 wherein R₁ is aminoand R₂ is hydrogen.
 4. A compound according to claim 1 wherein R₃ ishydroxymethyl.
 5. A compound according to claim 1 wherein R₅ and R₆ areboth hydrogen.
 6. A compound selected from the group consistingof:9-[2-(diethoxyphosphorylmethoxy)ethoxy]adenine,9-[2-(phosphonomethoxy)ethoxy]adenine,9-[2-(diethoxyphosphorylmethoxy)ethoxy]guanine,9-[2-(phosphonomethoxy)ethoxy]guanine,9-[2-[ethoxy(hydroxy)phosphorylmethoxy)ethoxy]guanine,9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]adenine,9-[3-hydroxy-2-(phosphonomethoxy)propoxy]adenine,9-[2-[ethoxy(hydroxy)phosphorylmethoxy]-3-hydroxypropoxy]adenine,9-[2-(diethoxyphosphorylmethoxy)-3-hydroxypropoxy]-guanine,9-[3-hydroxy-2-(phosphonomethoxy)propoxy]guanine,2,6-diamino-9-[2-(diethoxyphosphorylmethoxy)-ethoxy]purine,2,6-diamino-9-[2-phosphonomethoxy)ethoxy]purine,2-amino-6-chloro-9-[2-(phosphonomethoxy)ethoxy]purine,2-amino-6-methoxy-9-[2-(phosphonomethoxy)ethoxy]purine,9-[(2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)-methoxy]guanine,2,6-diamino-9-[3-hydroxy-2-(phosphonomethoxy)-propoxy]purine,(R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]guanine,(S)-9-[(2-hydroxy-2-oxo-1,4,2-dioxaphosphorinan-5-yl)-methoxy]guanine,(R)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]adenine,(S)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]guanine,(R)-9-[(2-hydroxy-2-oxo-1,4,2-dioxaphorphorinan-5-yl)methoxy]guanine,and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propoxy]adenine.
 7. A compoundaccording to claim 1, wherein R₃ is hydrogen.
 8. The compound9-[2-phosphonomethoxyethoxy]adenine.